A method for preparing an antibody-drug conjugate

Abstract

The present invention discloses a preparation method of an antibody-drug conjugate, including its synthesis and purification steps.

Description

[0001] This application claims priority to a Chinese patent application filed on March 25, 2020 (Application No. CN 202010219311.2) and a Chinese patent application filed on March 19, 2021 (Application No. CN 202110297397.5). Technical Field

[0002] This disclosure relates to a method for preparing a class of antibody-drug conjugates, and particularly to the synthesis and purification steps of a method for preparing a class of antibody-drug conjugates. Background Art

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

[0004] Chemotherapy remains one of the most important anti-cancer means, including surgery, radiotherapy, and targeted therapy. Although there are many types of highly effective cytotoxic drugs, the difference between tumor cells and normal cells is very small, limiting the wide clinical application of these anti-tumor compounds due to their toxic and side effects. Anti-tumor monoclonal antibodies are specific for tumor cell surface antigens, and antibody drugs have become front-line drugs for anti-tumor treatment. However, when antibodies are used alone as anti-tumor drugs, the efficacy is often unsatisfactory.

[0005] An antibody-drug conjugate (ADC) links a monoclonal antibody or antibody fragment to a bioactive cytotoxic drug through a stable chemical linker compound, making full use of the specificity of the antibody for binding to the surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic drug, while avoiding the defects of the low efficacy of the former and the excessive toxic and side effects of the latter. This means that, compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0006] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals, Inc.), was approved by the US FDA for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US 5079233; US 5585089; US 5606040; US5693762; US 5739116; US 5767285; US 5773001).

[0007] In August 2011, (brentuximab vedotin, Seattle Genetics, Inc.) was approved through the US FDA's accelerated approval pathway for the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma (Nat. Biotechnol(2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843; US7659241; WO2008025020). is a novel ADC drug that can directly act on the target CD30 on lymphoma cells and then undergo endocytosis to induce apoptosis of tumor cells.

[0008] Both Mylotarg and Adcetris are targeted therapies for hematological malignancies, which have relatively simple tissue structures compared to solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of patients with HER2-positive advanced or metastatic breast cancer who are resistant to both Tratuzumab (trade name: Herceptin) and paclitaxel (WO2005037992; US8088387). Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.

[0009] There are several types of cytotoxic small molecules for antibody-drug conjugates. One of them is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. The literature reporting the application of the camptothecin derivative irinotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) includes WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with better efficacy is still needed. SUMMARY OF THE INVENTION

[0010] 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 -Y-D):

[0011]

[0012] Wherein:

[0013] W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-7 cycloalkyl or a straight-chain heteroalkyl of 1 to 8 atoms, the straight-chain heteroalkyl containing 1 to 3 heteroatoms selected from N, O, and S, wherein the C 1-8 alkyl, C 3-7 cycloalkyl, and the straight-chain heteroalkyl are each independently optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, C 1-6 alkyl, chloro-C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy, and C 3-7 cycloalkyl;

[0014] L 2 is selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)-, and a chemical bond, where p 1 is an integer from 1 to 20;

[0015] L 3is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acid residues formed by amino acids in phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further selected from halogen, hydroxyl, cyano, amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 cycloalkyl; and is substituted by one or more substituents;

[0016] R 1 is halo C 1-6 alkyl or C 3-7 cycloalkyl;

[0017] R 2 is selected from a hydrogen atom, halo C 1-6 alkyl and C 3-7 cycloalkyl;

[0018] Alternatively, R 1 and R 2 together with the carbon atom to which they are attached form C 3-7 cycloalkyl;

[0019] R 5 is selected from a hydrogen atom, C 1-6 alkyl, halo C 1-6 alkyl, deuterated C 1-6 alkyl and hydroxy C 1-6 alkyl;

[0020] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, C 1-6 alkyl, halo C 1-6 alkyl, deuterated C 1-6 alkyl and hydroxy C 1-6 alkyl;

[0021] m is 0 or 1;

[0022] n is from 3 to 8, n is a decimal or an integer;

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

[0024] The preparation method includes the following steps:

[0025] Step (a): Reacting an antibody or an antigen-binding fragment thereof with a reducing agent at a reaction temperature of about 1 °C to about 36 °C;

[0026] Step (b): The product of step (a) is reacted with a compound represented by the following formula (La-Y-D);

[0027]

[0028] wherein: W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 and m are as defined above.

[0029] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate having a structure represented by the following formula:

[0030]

[0031] wherein, n is from 4 to 8, and n is a decimal or an integer;

[0032] The preparation method includes the following steps:

[0033] Step (a): The antibody or its antigen-binding fragment is reacted with a reducing agent at a reaction temperature of about 1 °C to about 36 °C;

[0034] Step (b): The product of step (a) is reacted with a compound represented by the following formula;

[0035]

[0036] In an alternative embodiment, the reaction temperature condition in step (a) is about 4 °C to about 30 °C, preferably about 20 °C to about 30 °C, more preferably 25 °C. Non-limiting examples include 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, about 30 °C. In some embodiments, the reaction temperature condition is 13 °C to 28 °C or 13 °C to 25 °C.

[0037] In an alternative embodiment, the reaction in step (a) is carried out under the condition that the pH is about 4.5 to about 6.5, preferably the reaction is carried out under the condition that the pH is about 5.0 to about 6.0, more preferably the reaction is carried out under the condition that the pH is 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.

[0038] In an alternative embodiment, the reaction in step (a) is carried out in a buffer; in a non-limiting example, the buffer is selected from histidine salt buffers, phosphate buffers, and acetate buffers.

[0039] In an alternative embodiment, the reaction in step (a) is carried out in a buffer; in a non-limiting example, the buffer is histidine-hydrochloride buffer.

[0040] In an alternative embodiment, the buffer is selected from histidine salt buffers containing EDTA and histidine-hydrochloride buffers containing EDTA. Exemplarily, the concentration of the histidine salt buffer is 1 mM to 100 mM, 10 mM to 50 mM, 20 mM, 30 mM, or 40 mM; the concentration of EDTA is 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 of the histidine salt buffer and 1 mM to 10 mM of EDTA. In some embodiments, the buffer contains 20 mM of the histidine-hydrochloride buffer and 2.5 mM of EDTA. EDTA refers to ethylenediaminetetraacetic acid.

[0041] In an alternative embodiment, the reducing agent in step (a) is selected from suitable reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) or its salts, 1,4-dithiothreitol (DTT), and β-mercaptoethanol (β-ME), preferably TCEP or its salts, more preferably tris(2-carboxyethyl)phosphine hydrochloride.

[0042] In an alternative embodiment, the molar ratio of the reducing agent to the antibody or its antigen-binding fragment (Pc) in step (a) is 2 to 10:1, 2.6 to 7:1, 2.9 to 3.7:1, 3.2 to 3.4:1, or 3.3:1.

[0043] In an alternative embodiment, step (b) is carried out in an organic solvent, and 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) includes: dissolving the compound represented by formula (La-Y-D) in DMSO and mixing the product of step (a) with the DMSO solution of the compound represented by formula (La-Y-D).

[0044] In an alternative embodiment, the above preparation method further comprises step (c), and step (c) comprises purifying the product of step (b). The purification can be carried out by cation exchange column chromatography or affinity column chromatography. The packing material for the cation exchange chromatography is selected from Capto S Impact and Poros XS, preferably Capto S Impact. In some embodiments, the Capto S Impact is Capto TM S Impact. In some embodiments, the Poros XS is Poros TM XS.

[0045] In an alternative embodiment, the drug loading (n) can range from 3 to 8, 4 to 8, 5 to 7, preferably 5.3 to 6.1, and more preferably 5.7 cytotoxic drugs per antibody or its antigen-binding fragment (Pc). n is a decimal or an integer. In some embodiments, n is 5.3, 5.4, 5.5, 5.6 or 5.7.

[0046] In an alternative embodiment, the drug loading distribution of the antibody-drug conjugate is as follows: in the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less; preferably, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 6% or less. In a non-limiting example, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, 3% or less, 2% or less, or 1% or less; the proportion of heavy chains of the antibody not conjugated with drugs is 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Alternatively, in the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 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 as follows: in the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: in the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: in the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less; and in the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: in the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less; and in the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 70% or more. In some embodiments, the proportion of the heavy chain of the antibody and / or the proportion of the light chain of the antibody are determined by reverse chromatography.

[0047] In an alternative embodiment, the preparation method is suitable for large-scale preparation. The input amount of trastuzumab in the 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.

[0048] In an alternative embodiment, the antibody-drug conjugate has a structure represented by the general formula (Pc-L b -Y-D):

[0049]

[0050] Wherein:

[0051] s 1 is an integer from 2 to 8;

[0052] Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as defined above;

[0053] The preparation method described above includes the following steps:

[0054] Step (a): React the antibody or its antigen-binding fragment with a reducing agent at a reaction temperature of about 1 to about 36 °C;

[0055] Step (b): React the product of step (a) with a compound represented by the following formula (L b -Y-D);

[0056]

[0057] where: s 1 , R 1 , R 2 , R 5 , R 6 , R 7 and m are as defined above.

[0058] In an alternative embodiment, the aforementioned antibody-drug conjugate has the following structure:

[0059]

[0060]

[0061]

[0062]

[0063] where Pc and n are as defined in the general formula (Pc-La-Y-D).

[0064] In an alternative embodiment, where the Pc is an antibody or its antigen-binding fragment, the antibody is selected from chimeric antibodies, humanized antibodies, and fully human antibodies. In some embodiments, the antibody is a monoclonal antibody.

[0065] In an alternative embodiment, the antibody or antigen-binding fragment thereof is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, and anti-Mesothelin antibody, or antigen-binding fragments thereof;

[0066] 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 antigen-binding fragments thereof.

[0067] In an alternative embodiment, the antibody conjugate has a structure represented by the following formula:

[0068]

[0069] wherein n is from 4 to 8, and n is a decimal or an integer.

[0070] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate having a structure represented by the following formula:

[0071]

[0072] wherein n is from 4 to 8, and n is a decimal or an integer;

[0073] The preparation method comprises the following steps:

[0074] Step (a): React 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;

[0075] Step (b): The product of step (a) is reacted with a compound represented by the following formula;

[0076]

[0077] In an alternative embodiment, the antibody-drug conjugate has a structure represented by the following formula:

[0078]

[0079] wherein n is from 4 to 8, and n is a fraction or an integer;

[0080] The preparation method comprises the following steps:

[0081] Step (a): Trastuzumab is reacted with TCEP at a reaction temperature of about 25 °C and a pH of about 5.6, and the reaction is carried out in a histidine-hydrochloric acid buffer containing EDTA;

[0082] Step (b): The product of step (a) is reacted with a compound represented by the following formula;

[0083]

[0084] Step (c): Comprises purifying the product of step (b) by cation exchange chromatography column or affinity chromatography column. In some embodiments, the histidine-hydrochloric acid buffer containing EDTA contains 20 mM histidine-hydrochloric acid buffer and 2.5 mM EDTA.

[0085] The present disclosure also 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 -Y-D):

[0086]

[0087] wherein:

[0088] W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-7 cycloalkyl or a straight-chain heteroalkyl of 1 to 8 atoms, the straight-chain heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8 alkyl, C 3-7 cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with a substituent selected from halogen, hydroxyl, cyano, amino, C 1-6 alkyl, chloro-C 1-6 alkyl, deuterated-C 1-6 alkyl, C 1-6 alkoxy and C 3-7substituted by one or more substituents in the cycloalkyl group;

[0089] L 2 selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- and chemical bonds, where p 1 is an integer from 1 to 20;

[0090] L 3 is a peptide residue composed of 2 to 7 amino acid residues, where the amino acid residues are selected from amino acid residues formed by amino acids in phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), aspartic acid (D), and are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-7 substituted by one or more substituents in the cycloalkyl group;

[0091] R 1 is halo C 1-6 alkyl or C 3-7 cycloalkyl;

[0092] R 2 is selected from a hydrogen atom, halo C 1-6 alkyl and C 3-7 cycloalkyl;

[0093] Alternatively, R 1 and R 2 together with the carbon atom to which they are attached form C 3-7 cycloalkyl;

[0094] R 5 is selected from a hydrogen atom, C 1-6 alkyl, halo C 1-6 alkyl, deuterated C 1-6 alkyl and hydroxy C 1-6 alkyl;

[0095] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, C 1-6 alkyl, halo C 1-6 alkyl, deuterated C 1-6 alkyl and hydroxy C 1-6 alkyl;

[0096] m is 0 or 1;

[0097] n is from 4 to 8, and n is a decimal or an integer;

[0098] Pc is an antibody or its antigen-binding fragment;

[0099] The drug load distribution of the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains bound to 4 drugs is 4% or less; preferably, the proportion of antibody heavy chains bound to 4 drugs is 4% or less, and the proportion of antibody heavy chains not bound to drugs is 6% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains bound to 4 drugs is 4% or less, and the proportion of antibody heavy chains not bound to drugs is 5% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains bound to 4 drugs is 1% or less, and the proportion of antibody heavy chains not bound to drugs is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains bound to 4 drugs is 4% or less, and the proportion of antibody heavy chains not bound to drugs is 5% or less; and in the population of antibody light chains, the proportion of antibody light chains bound to 1 drug is 65% or more. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains bound to 4 drugs is 1% or less, and the proportion of antibody heavy chains not bound to drugs is 4% or less; and in the population of antibody light chains, the proportion of antibody light chains bound to 1 drug is 70% or more.

[0100] The present disclosure also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is obtained by the method for preparing the antibody-drug conjugate described above; and, the drug loading distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less; preferably, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 6% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less; and among the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less; and among the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 70% or more.

[0101] The present disclosure also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by the following formula:

[0102]

[0103] wherein, n is from 4 to 8, and n is a decimal or an integer;

[0104] The antibody-drug conjugate is prepared by the method for preparing an antibody-drug conjugate as described above; and, the drug load distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less; preferably, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 6% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 4% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 5% or less; and among the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 65% or more. In some embodiments, the drug load distribution of the antibody-drug conjugate is as follows: among the heavy chain population of the antibody, the proportion of heavy chains of the antibody conjugated with 4 drugs is 1% or less, and the proportion of heavy chains of the antibody not conjugated with drugs is 4% or less; and among the light chain population of the antibody, the proportion of light chains of the antibody conjugated with 1 drug is 70% or more. Detailed Description of the Invention

[0106] The present disclosure provides a preparation method that is more conducive to large-scale production. Specifically, the product obtained by the preparation method has a narrower drug load distribution, a lower free toxin content, and a higher yield.

[0107] Terms

[0108] To facilitate a better understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains.

[0109] The present disclosure incorporates the entire content of Application PCT / CN2019 / 107873 into the present application.

[0110] "Antibody-drug conjugate (ADC)" refers to a conjugate that links an antibody or an antibody fragment to a bioactive cytotoxin or a small molecule drug with cell killing activity through a stable chemical linker compound, making full use of the specificity of the antibody for tumor cells or the specificity of binding to cells with highly expressed antigens and the high efficiency of the cytotoxin, and avoiding the toxic side effects on normal cells. Compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0111] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers for controlling pH within an appropriate range include acetate, succinate, gluconate, histidinate, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0112] "Histidinate buffer" is a buffer containing histidinate ions. Examples of histidinate buffers include buffers such as histidine-hydrochloric acid, histidine-acetate, histidine-phosphate, histidine-sulfuric acid, etc. The preferred histidinate buffer is histidine-hydrochloric acid buffer. The histidine-acetate buffer is prepared from histidine and acetic acid, and the histidine hydrochloride buffer is prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.

[0113] "Phosphate buffer" is a buffer including phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate - sodium dihydrogen phosphate, disodium hydrogen phosphate - potassium dihydrogen phosphate, disodium hydrogen phosphate - citric acid, etc. The preferred phosphate buffer is disodium hydrogen phosphate - sodium dihydrogen phosphate.

[0114] "Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include acetic acid - sodium acetate, histidine acetate, acetic acid - potassium acetate, calcium acetate acetate, acetic acid - magnesium acetate, etc. The preferred acetate buffer is acetic acid - sodium acetate.

[0115] As used herein, the terms "about" and "approximately" mean that a numerical value is within an acceptable error range of the specific value determined by those of ordinary skill in the art, and the numerical value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). For example, in each practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprises" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to one order of magnitude or up to 5 times the numerical value. Unless otherwise specified, when a specific value appears in this application and the claims, the meaning of "about" or "substantially comprises" should be assumed to be within the acceptable error range of that specific value.

[0116] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0117] As used herein, "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the difference in the constant region. Each of the five classes of Ig can have κ chains or λ chains. The antibody described in this disclosure is preferably a specific antibody against cell surface antigens on target cells. Non-limiting examples of such antibodies include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody, one or more of them; preferably trastuzumab (trastuzumab, trade name Herceptin), pertuzumab (pertuzumab, also known as 2C4, trade name Perjeta), nimotuzumab (nimotuzumab, trade name Tai Xinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, batimastat, gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.

[0118] The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly and are the variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions. The variable region includes 3 hypervariable regions (HVRs) and 4 relatively conserved framework regions (FRs). The 3 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 3 CDR regions and 4 FR regions, and the order arranged sequentially from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment described in this disclosure conform to the known Kabat numbering rules in terms of quantity and position (LCDR1-3, HCDR1-3).

[0119] In this disclosure, the antibody light chain described in this disclosure may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chains or variants thereof.

[0120] In this disclosure, the antibody heavy chain described in this disclosure may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0121] The antibodies of this disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

[0122] The term "murine antibody" in this disclosure refers to the preparation of antibodies in mice according to the knowledge and skills in the art. When preparing, a specific antigen is injected into the test subject, and then the hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.

[0123] The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, first establish a hybridoma secreting murine-specific monoclonal antibody, then clone the variable region gene from the murine hybridoma cells, then clone the constant region gene of the human antibody as needed, connect the murine variable region gene with the human constant region gene to form a chimeric gene and insert it into a human vector, and finally express the chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system.

[0124] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by transplanting the CDR sequences of a mouse into the framework of the variable region of a human antibody, i.e., into different types of human germline antibody framework sequences. It can overcome the strong heterologous reactions induced by chimeric antibodies due to the presence of a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" germline sequence database and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity while minimizing immunogenicity, the human antibody variable region framework sequences can be subjected to minimal back mutations or revertant mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further affinity matured by phage display of the CDRs.

[0125] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (e.g., a cytotoxic module) or a radiolabel.

[0126] The "antigen-binding fragment of an antibody" as described in this disclosure may refer to Fab fragments, Fab' fragments, F(ab')2 fragments having antigen-binding activity, and Fv fragments, scFv fragments that bind to an antigen. The Fv fragment contains the variable regions of the heavy and light chains of an antibody but no constant regions and is the smallest antibody fragment having all the antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, referred to as single chain antibody or single chain Fv (sFv).

[0127] The term "antigen-binding site" as used in this disclosure refers to a three-dimensional site on an antigen, either continuous or discontinuous, recognized by the antibodies or antigen-binding fragments disclosed herein.

[0128] "ADCC" as described in this disclosure, namely antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognizing the Fc segment of the antibody. The ADCC effector function of the antibody can be reduced or eliminated by modifying the Fc segment on IgG. The modification refers to mutating the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimera, F234A / L235A mutation of IgG4.

[0129] "Mutation" in the mutant sequences described in this disclosure includes but is not limited to "reverse mutation", "conservative modification" or "conservative substitution or replacement". "Conservative modification" or "conservative substitution or replacement" as described in this disclosure refers to the substitution of an amino acid in a protein by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), so that the change can be carried out frequently without changing the biological activity of the protein. Those skilled in the art know that generally, a single amino acid substitution in a non-essential region of a polypeptide basically does not change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). In addition, the substitution of amino acids with similar structures or functions is unlikely to destroy the biological activity.

[0130] The "mutant sequence" described in this disclosure refers to a nucleotide sequence and / or amino acid sequence obtained by performing appropriate mutation modifications such as substitution, insertion or deletion on the nucleotide sequence and / or amino acid sequence of this disclosure, and having a different percentage sequence identity degree with the nucleotide sequence and / or amino acid sequence of this disclosure. The sequence identity described in this disclosure can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The sequence comparison and determination of the percentage identity between two sequences can be carried out by the default settings of the BLASTN / BLASTP algorithm available on the website of the National Center For Biotechnology Institute.

[0131] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to an antibody or a drug after connecting to other linkers.

[0132] Linkers, including spacers, linkers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates the release of the drug in the cell. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0133] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the 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. As a more recommended prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in a serum-free medium in a bioreactor to produce the antibody. The culture broth secreting the antibody can be purified by conventional techniques. For example, purification can be performed using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieving and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0134] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and their various branched isomers, etc. More preferably, it is a lower alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available attachment point. The substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo group.

[0135] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, and S, wherein the alkyl group is as defined above.

[0136] The term "alkylene" refers to a saturated straight-chain 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 the parent alkane, which is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylethylene (-CH(CH3)-), 1,2-ethylethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), etc. The alkylene may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably independently optionally selected from one or more substituents of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo group.

[0137] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where the alkyl or cycloalkyl is defined as above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0138] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, and the cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 12 carbon atoms, more preferably contains 3 to 10 carbon atoms, and most preferably contains 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyl includes spirocyclic, fused-ring, and bridged-ring cycloalkyl.

[0139] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which contains 3 to 20 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O) mheteroatoms (where m is an integer from 0 to 2), but excluding ring moieties of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, among which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

[0140] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members in which a single atom (called the spiro atom) is shared between monocyclic rings, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m heteroatoms (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π - electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Spiroheterocyclic groups are classified into monospiroheterocyclic groups, dispiroheterocyclic groups or polyspiroheterocyclic groups according to the number of spiro atoms shared between rings, and monospiroheterocyclic groups and dispiroheterocyclic groups are preferred. More preferably, they are 4 - member / 4 - member, 4 - member / 5 - member, 4 - member / 6 - member, 5 - member / 5 - member or 5 - member / 6 - member monospiroheterocyclic groups. Non - limiting examples of spiroheterocyclic groups include:

[0141]

[0142] The term "fused heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated π - electron system, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m heteroatoms (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, and bicyclic or tricyclic groups are preferred, and 5 - member / 5 - member or 5 - member / 6 - member bicyclic fused heterocyclic groups are more preferred. Non - limiting examples of fused heterocyclic groups include:

[0143]

[0144] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 14 members in which any two rings share two non - directly - connected atoms, and it may contain one or more double bonds, but none of the rings has a fully conjugated π - electron system, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) ma heteroatom (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 membered, more preferably 7 to 10 membered. It can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups according to the number of rings forming the cycle, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of the bridged heterocyclic group include:

[0145]

[0146] The heterocyclic group ring can be fused to an aryl, heteroaryl or cycloalkyl ring, where the ring connected to the parent structure is the heterocyclic group, and non-limiting examples thereof include:

[0147] etc.

[0148] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo group.

[0149] The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring can be fused to a heteroaryl, heterocyclic or cycloalkyl ring, where the ring connected to the parent structure is the aryl ring, and non-limiting examples thereof include:

[0150]

[0151] The aryl can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

[0152] The term "heteroaryl" 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. The heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring, and non-limiting examples thereof include:

[0153]

[0154] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

[0155] The term "amino protecting group" is used to protect the amino group with a group that can be easily removed so that the amino group remains unchanged when other parts of the molecule are reacted. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, etc. These groups may optionally be substituted with 1-3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

[0156] The term "cycloalkylalkyl" means that the alkyl group is substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the alkyl group is as defined above and the cycloalkyl group is as defined above.

[0157] The term "haloalkyl" means that the alkyl group is substituted with one or more halogen atoms, wherein the alkyl group is as defined above.

[0158] The term "deuterated alkyl" means that the alkyl group is substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0159] The term "hydroxy" refers to the -OH group.

[0160] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0161] The term "amino" refers to -NH2.

[0162] The term "nitro" refers to -NO2.

[0163] The term "cyano" refers to -CN.

[0164] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and this description includes the cases where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may, but need not, be present.

[0165] "Substituted" means that one or more hydrogen atoms in the group, preferably up to 5, more preferably 1-3 hydrogen atoms, are independently replaced by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) what substitutions are possible or impossible without undue effort. For example, an amino or hydroxy group with a free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (such as olefinic) bond.

[0166] The term "drug - loading amount" or "drug - to - antibody ratio" (DAR) refers to the average number of cytotoxic drugs loaded on each antibody or its antigen - binding fragment in an ADC, and can also be expressed as the ratio of the amount of drug to the amount of antibody, which is an integer or a decimal. In the embodiments of the present disclosure, the drug - loading amount is denoted as n, and can exemplarily be the average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0167] The term "drug - loading distribution" refers to the distribution of antibodies conjugated with different numbers of drugs in a population of antibody - drug conjugates. For example, the distribution of antibodies conjugated with 0, 2, 4, 6, and 8 drugs in the population. It should be noted that due to the possible generation of degradation products, DARs of 1, 3, 5, and 7 may also be included in the mixture. In the present disclosure, the antibody - drug loading distribution can be characterized by heavy chains of antibodies conjugated with different numbers of drugs. For example: H0 represents the heavy chain not conjugated with drugs, H1 represents the heavy chain conjugated with one drug, H2 represents the heavy chain conjugated with two drugs, H3 represents the heavy chain conjugated with three drugs, and H4 represents the heavy chain conjugated with four drugs. Exemplarily, an H3 ratio of 4% means that in the heavy - chain population of the antibody - drug conjugate, the proportion of heavy chains conjugated with three drugs is 4%. Correspondingly, in the present disclosure, the antibody - drug loading distribution can also be characterized by light chains of antibodies conjugated with different numbers of drugs. L0 represents the light chain of the antibody not conjugated with drugs, and L1 represents the light chain of the antibody conjugated with 1 drug.

[0168] "Administer", "administered", and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administer", "administered", and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of cells includes the contact of a reagent with the cells and the contact of the reagent with a fluid that contacts the cells. "Administer", "administered", and "treatment" also mean the treatment of cells in vitro and ex vivo by a reagent, diagnostic, binding composition, or by another cell. "Treatment" when applied to humans, veterinary medicine, or research subjects refers to therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[0169] "Treatment" means administering to a patient a therapeutic agent, either internally or externally, such as a composition comprising any of the binding compounds disclosed herein, to a patient having one or more disease symptoms, where the therapeutic agent is known to have a therapeutic effect on such symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to relieve one or more disease symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to relieve any specific disease symptom (also referred to as the "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient. Whether a disease symptom has been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare provider to evaluate the severity or progression of the symptom. Although the embodiments disclosed herein (such as treatment methods or articles) may not be effective in relieving each target disease symptom, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0170] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as, for example, the condition to be treated, the overall health of the patient, the route and dosage of administration, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0171] "Replacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, the high-salt or hypertonic solvent system containing the antibody protein is replaced by a buffer system of a stable formulation through physical manipulation means, so that the antibody protein is present in the stable formulation. The so-called physical manipulation means include, but are not limited to, ultrafiltration, dialysis, or redissolution after centrifugation. Description of the Drawings

[0172] Figure 1A : Results of the plasma stability experiment of ADC-19 disclosed herein.

[0173] Figure 1B : Results of the plasma stability experiment of ADC-18 disclosed herein.

[0174] Figure 1C : Results of the plasma stability experiment of ADC-20 disclosed herein.

[0175] Figure 2: This invention discloses the efficacy evaluation of ADC-21 and ADC-24 on JIMT-1 tumor-bearing mice.

[0176] Figure 3 :The efficacy of the disclosed ADC on nude mice transplanted with human breast cancer cells SK-BR-3 was evaluated.

[0177] Figure 4 : The present invention discloses the plasma stability experimental results of ADC-25.

[0178] Figure 5 :The present invention discloses the efficacy of ADC on human brain astrocytoma U87MG nude mouse transplanted tumors.

[0179] Figure 6 :The present invention discloses the efficacy of ADC on Detroit 562 nude mouse transplanted tumors of human pharyngeal carcinoma pleural effusion metastatic cells.

[0180] Figure 7 :The present invention discloses the efficacy of ADC on human glioma U87MG nude mouse transplanted tumors. DETAILED DESCRIPTION

[0181] The present disclosure is further described and explained below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

[0182] The experimental methods in the examples disclosed herein that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0183] 1. Preparation, properties and biological testing of antibody-drug conjugates

[0184] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR was measured using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) as the solvent, tetramethylsilane (TMS) as the internal standard, and chemical shifts at 10 -6 (ppm) is given as the unit.

[0185] MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

[0186] UPLC was measured using a Waters Acquity UPLC SQD liquid spectrometer.

[0187] The determination by HPLC was performed using an Agilent 1200 DAD high-pressure liquid chromatograph (Sunfire C18 150×4.6 mm chromatographic column) and a Waters 2695-2996 high-pressure liquid chromatograph (Gimini C18 150×4.6 mm chromatographic column).

[0188] The determination by UV-HPLC was performed using a Thermo nanodrop2000 ultraviolet spectrophotometer.

[0189] Proliferation inhibition rate and IC 50 value were determined using a PHERA star FS microplate reader (BMG Labtech, Germany).

[0190] For thin-layer chromatography silica gel plates, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates were used. The specifications of the silica gel plates used in thin-layer chromatography (TLC) were 0.15 mm - 0.2 mm, and the specifications of the silica gel plates used for thin-layer chromatography separation and purification of products were 0.4 mm - 0.5 mm.

[0191] For column chromatography, silica gel with 200 - 300 mesh from Yantai Huanghai was generally used as the carrier.

[0192] The known starting materials of this disclosure can be synthesized by adopting or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0193] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

[0194] An argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.

[0195] A hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L.

[0196] The catalytic hydrogenation reaction was carried out using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0197] For the hydrogenation reaction, it was usually evacuated, filled with hydrogen, and this operation was repeated 3 times.

[0198] The microwave reaction was carried out using a CEM Discover-S 908860 microwave reactor.

[0199] Unless otherwise specified in the examples, the solutions in the reactions refer to aqueous solutions.

[0200] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0201] Room temperature is the most suitable reaction temperature, and the temperature range is 20°C to 30°C.

[0202] Preparation of PBS buffer solution with pH = 6.5 in the examples: Weigh 8.5 g of KH2PO4, 8.56 g of K2HPO4·3H2O, 5.85 g of NaCl, and 1.5 g of EDTA and place them in a flask. Make up the volume to 2 L, dissolve all of them by ultrasonic wave, and shake well to obtain it.

[0203] The eluent system for column chromatography and the developing agent system for thin-layer chromatography used for purifying the compound include: A: dichloromethane and isopropanol system; B: dichloromethane and methanol system; C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or basic reagents can also be added for adjustment.

[0204] Some compounds in this disclosure are characterized by Q-TOF LC / MS. Q-TOF LC / MS uses 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 5μm, 2.1×75mm chromatographic column).

[0205] Example 1-1

[0206] 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

[0207]

[0208] To irinotecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), 1 mL of N,N-dimethylformamide was added, and the mixture was cooled to 0 - 5 °C in an ice-water bath. A drop of triethylamine was added, and the mixture was stirred until the reaction solution became clear. 1-Hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by the well-known method "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 successively added to the reaction solution. After addition, the mixture was stirred at 0 - 5 °C for 2 hours. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 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 eluent system B to obtain the title product 1 (1.6 mg, yield: 82.1%).

[0209] MS m / z(ESI): 520.2[M + 1]

[0210] 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).

[0211] Example 1 - 2

[0212] (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

[0213] (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

[0214]

[0215] To 1b (4 mg, 7.53 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added. After three replacements with argon, the mixture was cooled to 0 - 5 °C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise, followed by stirring until the reaction solution became clear. To the reaction solution were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared by the method disclosed in the patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After addition, 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 solution was concentrated under reduced pressure, and the resulting crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridgePrep 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 fractions were collected and concentrated under reduced pressure to obtain the title products (2-A: 1.5 mg, 2-B: 1.5 mg).

[0216] MS m / z (ESI): 534.0 [M+1].

[0217] Single configuration compound 2-B (shorter retention time)

[0218] UPLC analysis: retention time 1.06 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0219] 11H 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).

[0220] Single - configuration compound 2 - A (longer retention time)

[0221] UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEH C18 1.7 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0222] 1 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).

[0223] Examples 1 - 3

[0224] (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

[0225] (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

[0226]

[0227] To 1b (5.0 mg, 9.41 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, and the mixture was cooled to 0 - 5 °C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. To the reaction solution were successively added 3,3,3-trifluoro-2-hydroxypropanoic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol). After the addition, 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 solution was concentrated under reduced pressure, and the resulting crude 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 fraction was collected and concentrated under reduced pressure to obtain the title product (1.5 mg, 1.5 mg).

[0228] MS m / z (ESI): 561.9 [M + 1].

[0229] Single configuration compound (shorter retention time)

[0230] UPLC analysis: retention time 1.11 minutes, purity: 88% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0231] 11H 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).

[0232] Single-configuration compound (longer retention time)

[0233] UPLC analysis: retention time 1.19 minutes, purity: 90% (column: ACQUITY UPLC BEH C18 1.7 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0234] 1 1H 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).

[0235] Examples 1 - 4

[0236] 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

[0237]

[0238] 1b (3.0 mg, 5.64 μmol) was added to 1 mL of N,N-dimethylformamide, cooled to 0 - 5 °C in an ice-water bath, and one drop of triethylamine was added dropwise. The mixture was stirred until the reaction solution became clear. 1-Hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared by the method disclosed in the patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were successively added to the reaction solution. After addition, the reaction was stirred at 0 - 5 °C for 1 hour. 5 mL of water was added to the reaction solution to quench the reaction. The reaction solution was extracted with ethyl acetate (10 mL × 3), and the organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing agent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).

[0239] MS m / z (ESI): 548.0 [M + 1].

[0240] 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).

[0241] Examples 1 - 5

[0242] 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

[0243]

[0244] 1b (2.0 mg, 3.76 μmol) was added to 1 mL of N,N-dimethylformamide, cooled to 0 - 5 °C in an ice-water bath, and a drop of triethylamine was added dropwise, followed by stirring until the reaction solution became clear. 1-(Hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared by the method disclosed in the patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were successively added to the reaction solution. After addition, the reaction was stirred at 0 - 5 °C for 2 hours. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 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 the developing agent system B to obtain the title product 5 (1.0 mg, yield: 50%).

[0245] MS m / z(ESI): 533.9[M + 1].

[0246] 1 H 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).

[0247] Examples 1 - 6

[0248] 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

[0249]

[0250] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), and the mixture was cooled to 0 - 5 °C in an ice-water bath. One drop of triethylamine was added dropwise, and the mixture was stirred until the reaction solution became clear. 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared by the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, p. 8138 - 8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were successively added to the reaction solution. After addition, the mixture was stirred at 0 - 5 °C for 1 hour. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by thin-layer chromatography with eluent system B to obtain the title product 6 (2.1 mg, yield: 67.9%).

[0251] MS m / z (ESI): 548.0 [M + 1].

[0252] 1 1H 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).

[0253] Examples 1 - 7

[0254] 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

[0255]

[0256] To 1b (3.0 mg, 5.64 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added. It was cooled to 0 - 5 °C in an ice-water bath, 0.3 mL of N-methylmorpholine was added dropwise, and stirred until the reaction solution became clear. To the reaction solution were successively added 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplied by Yoshi Pharma), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol). After addition, the reaction was stirred at 0 - 5 °C for 10 minutes. The ice-water bath was removed, and the reaction was stirred at room temperature for 2 hours. The reaction solution 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%).

[0257] MS m / z (ESI): 534.0 [M+1].

[0258] 1 1H 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).

[0259] Example 1 - 8

[0260] 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-pentaazatetracosanyl)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

[0261]

[0262]

[0263] The first step

[0264] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

[0265] 1-Hydroxybenzyl cyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol; prepared by the method disclosed in the 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 the patent application "CN105829346A") were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, and the mixture was stirred at room temperature for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to obtain the title product 8c (100 mg, yield: 73.6%).

[0266] MS m / z (ESI): 501.0 [M + 1].

[0267] Second step

[0268] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0269] 8c (50 mg, 0.10 mmol) was dissolved in a mixed solvent of 3 mL of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium on carbon (25 mg, 10% content) was added, the mixture was purged with hydrogen three times, and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the title product 8d (41 mg, yield: 100%).

[0270] MS m / z (ESI): 411.0 [M + 1].

[0271] Third step

[0272] (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

[0273] Add 1b (7 mg, 0.013 mmol) to a reaction flask, add 1 mL of N,N-dimethylformamide, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add a drop of triethylamine, add a 0.5 mL N,N-dimethylformamide solution of 8d (7 mg, 0.017 mmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol), and stir the reaction in an ice bath for 35 minutes. Add 10 mL of water, extract with ethyl acetate (5 mL × 3), wash the organic phase with saturated sodium chloride solution (10 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the resulting residue by thin-layer chromatography using eluent system B to obtain the title product 8e (8.5 mg, yield 78.0%).

[0274] MS m / z (ESI): 828.0 [M+1].

[0275] The fourth step

[0276] 1-((2-Aminoacetamido)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

[0277] Dissolve 8e (4 mg, 4.84 μmol) in 0.2 mL of dichloromethane, add 0.1 mL of diethylamine, and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure twice, add 3 mL of n-hexane to slurry, pour out the upper n-hexane layer, repeat three times, and concentrate under reduced pressure to obtain the crude title product 8f (2.9 mg), which is directly used in the next step without purification.

[0278] MS m / z (ESI): 606.0 [M+1].

[0279] The fifth step

[0280] 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-pentaazaeicosanyloxy)-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

[0281] Dissolve the crude product 8f (2.9 mg, 4.84 μmol) in 0.5 mL of N,N-dimethylformamide. Replace the gas with argon three times. Cool the solution to 0 - 5 °C in an ice-water bath. Add a 0.3 mL N,N-dimethylformamide solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetamido)acetamido)-3-phenylpropanoic acid 8g (2.7 mg, 5.80 μmol, prepared by the method disclosed in the patent application "EP2907824"). Add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). Stir the reaction mixture in an ice bath for 30 minutes. Remove the ice bath and stir at room temperature for 15 minutes. Purify the reaction mixture by high-performance liquid chromatography (separation conditions: chromatographic 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). Collect the corresponding fractions and concentrate under reduced pressure to obtain the title product 8 (2 mg, yield: 39.0%).

[0282] MS m / z (ESI): 1060.0 [M+1].

[0283] 11H 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).

[0284] Examples 1 - 9

[0285] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0286] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0287]

[0288]

[0289] The first step

[0290] Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0291] 2a (1.3 g, 11.2 mmol; prepared by the method disclosed in the patent application "WO2013 / 106717") was dissolved in 50 mL of acetonitrile. 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 successively. 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, and the residue obtained was purified by silica gel column chromatography with eluent system C to give the title product 9a (2 g, yield: 86.9%).

[0292] The second step

[0293] Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oate 9b

[0294] 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, and 4 mL of tetrahydrofuran was added. The reaction system was purged with argon three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice bath was removed, and the mixture was stirred at room temperature for 40 minutes. Then 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 phases were concentrated. The residue obtained was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography with eluent system C to give the title product 9b (48 mg, yield: 19%).

[0295] MS m / z (ESI): 515.0 [M+1].

[0296] The third step

[0297] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oic acid 9c

[0298] 9b (20 mg, 0.038 mmol) was dissolved in a mixed solvent of 4.5 mL of tetrahydrofuran and ethyl acetate (V:V = 2:1). Palladium on carbon (12 mg, 10% content, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth. The filter cake was rinsed with ethyl acetate. The filtrate was concentrated to obtain the crude title product 9c (13 mg). The product was directly used for the next reaction without purification.

[0299] MS m / z (ESI): 424.9 [M+1].

[0300] The fourth step

[0301] (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

[0302] 1b (10 mg, 18.8 μmol) was added to a reaction flask. 1 mL of N,N-dimethylformamide was added. The mixture was purged with argon three times and cooled to 0 - 5 °C in an ice-water bath. One drop of triethylamine was added. The crude 9c (13 mg, 30.6 μmol) was added. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) was added. The mixture was stirred at ice-bath temperature for 40 minutes. 10 mL of water was added. The mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The combined organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by thin-layer chromatography using eluent system B to obtain the title product 9d (19 mg, yield: 73.6%).

[0303] MS m / z (ESI): 842.1 [M+1].

[0304] The fifth step

[0305] 2-((2-Aminoacetamido)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

[0306] Dissolve 9d (19 mg, 22.6 μmol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, add 1 mL of toluene and concentrate under reduced pressure again, repeating twice. Add 3 mL of n-hexane to the residue for pulping, let it stand and pour out the supernatant, retaining the solid. Concentrate the solid residue under reduced pressure and dry it with an oil pump to obtain the crude title product 9e (17 mg), and the product is directly used for the next reaction without purification.

[0307] MS m / z(ESI): 638.0[M+18].

[0308] Step 6

[0309] N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0310] N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0311] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0 - 5 °C in an ice-water bath. Then, 8 g (21.2 mg, 44.8 μmol) of 0.3 mL N,N-dimethylformamide solution was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The reaction mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed and the mixture was stirred at room temperature for 1 hour to form compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A - water (10 mmol NH4OAc): B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding fractions were collected, concentrated under reduced pressure to obtain the title products (9-A: 2.4 mg, 9-B: 1.7 mg).

[0312] MS m / z(ESI): 1074.4[M + 1].

[0313] Single configuration compound 9-A (shorter retention time):

[0314] UPLC analysis: retention time 1.14 minutes, purity: 85% (column: ACQUITY UPLC BEH C18 1.7μm 2.1*50mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0315] 11H 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).

[0316] Single - configuration compound 9 - B (longer retention time):

[0317] UPLC analysis: retention time 1.16 minutes, purity: 89% (column: ACQUITY UPLC BEH C18 1.7 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0318] 11H 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).

[0319] Examples 1 - 10

[0320] 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-tetraazapentadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A

[0321] 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-tetraazapentadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0322]

[0323]

[0324] The first step

[0325] Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

[0326] Dissolve 3a (1.80 g, 12.5 mmol) in 100 mL of acetonitrile, and successively add potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol) and tetrabutylammonium iodide (231 mg, 0.63 mmol). Heat the reaction solution to 60 °C and stir for 5 hours. Cool the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure, and purify the obtained residue by silica gel column chromatography with eluent system C to obtain the title product 10a (980 mg, yield: 33.5%).

[0327] 1 1H NMR (400 MHz, CDCl3): δ 7.43 - 7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

[0328] The second step

[0329] Benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazoundec-11-oate 10b

[0330] Add 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) to a reaction flask, add 3 mL of tetrahydrofuran, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add potassium tert-butoxide (38 mg, 0.34 mmol), remove the ice bath, warm to room temperature and stir for 20 minutes, add 10 mL of ice water, extract with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5), combine the organic phases and concentrate. Dissolve the obtained residue in 2 mL of dioxane, add 0.4 mL of water, add sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol), stir at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained residue by silica gel column chromatography with eluent system C to obtain the title product 10b (51 mg, yield: 55.3%).

[0331] MS m / z (ESI): 559.9 [M + 18].

[0332] The third step

[0333] 1-(9H-Fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazoundec-11-oic acid 10c

[0334] Dissolve 10b (15 mg, 0.28 mmol) in a mixed solvent of 3 mL of tetrahydrofuran and ethyl acetate (V:V = 2:1), add palladium on carbon (15 mg, 10% content), displace with hydrogen three times, and stir the reaction at room temperature for 1 hour. Filter the reaction solution through diatomaceous earth, wash the filter cake with tetrahydrofuran, concentrate the filtrate to obtain the crude title product 10c (13 mg).

[0335] MS m / z (ESI): 452.9 [M+1].

[0336] The fourth step

[0337] (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

[0338] Add 1b (10 mg, 18.8 μmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add a drop of triethylamine, add a 0.5 mL N,N-dimethylformamide solution of 10c (13 mg, 28.7 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (11 mg, 39.7 μmol), and stir the reaction in an ice bath for 30 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), combine the organic phases, wash the organic phases with saturated sodium chloride solution (10 mL × 2), dry the organic phases with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the resulting residue by thin-layer chromatography with eluent system B to obtain the title product 10d (16 mg, yield 97.8%).

[0339] MS m / z (ESI): 870.0 [M+1].

[0340] The fifth step

[0341] 2-((2-Aminoacetamido)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

[0342] Dissolve 10d (16 mg, 18.4 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 2 hours. Concentrate the reaction mixture under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure again, repeating twice. Add 3 mL of n-hexane to the residue to form a slurry, let it stand, and then pour out the supernatant liquid, retaining the solid; repeat three times. Concentrate the solid residue under reduced pressure and dry it with an oil pump to obtain the crude title product 10e (12 mg). The product is used directly in the next reaction without purification.

[0343] MS m / z (ESI): 647.9 [M+1].

[0344] The Sixth Step

[0345] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A

[0346] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0347] The crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0 - 5 °C in an ice-water bath. An 8 g (14 mg, 29.6 μmol) solution 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 (15 mg, 54.2 μmol). The reaction mixture was stirred in the ice bath for 30 minutes, then the ice bath was removed and the mixture was stirred at room temperature for 1 hour to form Compound 10. The reaction solution 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 fractions were collected, concentrated under reduced pressure to obtain the title product (2.7 mg, 2.6 mg).

[0348] MS m / z (ESI): 1102.0 [M+1].

[0349] Single configuration compound (shorter retention time):

[0350] UPLC analysis: retention time 1.18 minutes, purity: 91% (column: ACQUITY UPLC BEH C18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0351] 1 H 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).

[0352] Single-configuration compound (longer retention time):

[0353] UPLC analysis: retention time 1.23 minutes, purity: 90% (column: ACQUITY UPLC BEH C18 1.7μm 2.1*50mm, mobile phase: A - water (5mmol NH4OAc), B - acetonitrile).

[0354] 1 H NMR (400MHz, DMSO-d6): δ9.05 (d, 1H), 8.97 - 8.88 (m, 1H), 8.35 - 8.27 (m, 1H), 8.11 - 8.03 (m, 1H), 8.02 - 7.95 (m, 1H), 7.80 (d, 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).

[0355] Example 1 - 11

[0356] 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 - pentaazadocosanyl)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

[0357]

[0358]

[0359] The first step

[0360] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylate 11b

[0361] 1-Hydroxycyclobutane-carboxylic acid benzyl ester 11a (167 mg, 0.81 mmol, prepared by the method disclosed in the literature "Journal of Medicinal Chemistry, 2013, vol. 56, #13, p. 5541 - 5552") and 8b (150 mg, 0.41 mmol) were added to a reaction flask, 5 mL of tetrahydrofuran was added, and the mixture was purged with argon 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, and the mixture was stirred at room temperature for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 mL of dioxane, 0.6 mL of water was added, sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 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 with eluent system C to obtain the title product 11b (37 mg, yield: 17.6%).

[0362] MS m / z (ESI): 514.6 [M + 1].

[0363] The second step

[0364] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11c

[0365] 11b (37 mg, 71.9 μmol) was dissolved in a mixed solvent of 3 mL 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 three times. The mixture was stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the title product 11c (35 mg, yield: 82%), which was directly used in the next step.

[0366] The third step

[0367] (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)cyclobutoxy)methyl)amino)-2-oxoethyl)carbamate 11d

[0368] Add 1b (10 mg, 0.018 mmol) to a reaction flask, add 1 mL of N,N-dimethylformamide, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add a drop of triethylamine, add a 0.5 mL N,N-dimethylformamide solution of 11c (13 mg, 0.031 mmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol), and stir the reaction in an ice bath for 40 minutes. Add 8 mL of water, extract with ethyl acetate (5 mL × 3), wash the organic phase with saturated sodium chloride solution (8 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the resulting residue by thin-layer chromatography using eluent system A to obtain the title product 11d (19 mg, yield 73.9%).

[0369] MS m / z (ESI): 842.3 [M + 1].

[0370] The fourth step

[0371] 1-((2-Aminoacetamido)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

[0372] Dissolve 11d (19 mg, 22.6 μmol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 1.5 hours. Concentrate the reaction solution under reduced pressure, add 1 mL of toluene and concentrate under reduced pressure twice, add 4 mL of n-hexane to slurry, pour out the upper layer of n-hexane three times, and concentrate under reduced pressure to obtain the crude title product 11e (15 mg), and the product is directly used in the next step without purification.

[0373] The fifth step

[0374] 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-pentaazadocosan-1-yl)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

[0375] Dissolve the crude product 11e (2 mg, 3.22 μmol) in 0.5 mL of N,N-dimethylformamide, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add 8 g (1.5 mg, 3.17 μmol) of its 0.3 mL N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol), and stir at room temperature for 30 minutes. Rotavaporize the reaction mixture to dryness with an oil pump to remove DMF, dissolve the residue in DCM and directly purify it by thin-layer chromatography 2 times (developing agent polarity: DCM / MeOH = 10 / 1) to obtain the title product 11 (1 mg, yield: 28.8%).

[0376] MS m / z (ESI): 1073.6 [M+1].

[0377] 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).

[0378] Example 1 - 12

[0379] (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

[0380] (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

[0381]

[0382] The first step

[0383] 3-Cyclopropyl-2-hydroxypropanoic acid 12b

[0384] Dissolve 12a (0.5 g, 3.87 mmol, supplier Adamas) in a mixed solvent of 35 mL of water and acetic acid (V:V = 4:1), cool the solution to 0 - 5 °C in an ice-water bath, dropwise add a 2 M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol), raise the temperature to room temperature and stir the reaction for 3 hours. Add solid sodium chloride to the reaction solution to saturate the aqueous phase, extract with ethyl acetate (8 mL × 8), dry over anhydrous sodium sulfate, filter, and concentrate to obtain the title product 12b (0.45 g, yield: 89.3%).

[0385] The second step

[0386] (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

[0387] (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

[0388] 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide were added to 1b (45 mg, 0.085 mmol). The mixture was purged with argon three times, and 0.1 mL of N-methylmorpholine was added dropwise. The mixture was stirred until the reaction solution 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 successively added to the reaction solution. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatographic 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 obtain the title product (7 mg, 15 mg).

[0389] MS m / z (ESI): 547.9 [M+1].

[0390] Single configuration compound (shorter retention time)

[0391] UPLC analysis: retention time 1.345 minutes, purity: 72% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0392] 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).

[0393] Single configuration compound (longer retention time)

[0394] UPLC analysis: retention time 1.399 minutes, purity: 88% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0395] 1 1H 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).

[0396] Examples 1 - 13 (Reference Examples)

[0397] 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

[0398]

[0399] The title compound 13 was prepared according to the method disclosed in "Example 76 on page 147 of the specification of Patent 'EP2907824A1'".

[0400] Examples 1 - 14

[0401] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14 - A

[0402] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0403]

[0404]

[0405] The first step

[0406] Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0407] Dissolve 12b (200 mg, 1.54 mmol) in 20 mL of acetonitrile, and successively add potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol) and tetrabutylammonium iodide (28 mg, 0.07 mmol). Stir the reaction mixture at room temperature for 48 hours, filter it through diatomaceous earth, wash the filter cake with ethyl acetate (10 mL), combine the filtrates and concentrate them under reduced pressure. Purify the obtained residue by silica gel column chromatography with eluent system C to obtain the title product 14a (140 mg, yield: 41.3%).

[0408] The second step

[0409] Benzyl 10-(cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazoundec-11-oate 14b

[0410] Add 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) to a reaction flask, add 10 mL of tetrahydrofuran, displace the air with argon three times, cool the reaction mixture to 0 - 5 °C in an ice-water bath, add potassium tert-butoxide (79 mg, 0.704 mmol), remove the ice bath, warm the reaction mixture to room temperature and stir for 10 minutes, add 20 mL of ice water, extract with ethyl acetate (10 mL × 4), wash the organic phase with saturated sodium chloride solution (20 mL), dry it over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by silica gel column chromatography with eluent system C to obtain the title product 14b (50 mg, yield: 26.8%).

[0411] MS m / z (ESI): 529.2 [M+1].

[0412] The third step

[0413] 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oic acid 14c

[0414] Dissolve 14b (27 mg, 0.051 mmol) in 3 mL of ethyl acetate, add palladium on carbon (7 mg, 10% content, dry type), displace with hydrogen three times, and stir the reaction at room temperature for 1 hour. Filter the reaction solution through diatomaceous earth, wash the filter cake with ethyl acetate, concentrate the filtrate to obtain the crude title product 14c (23 mg), and directly carry out the next reaction without purifying the product.

[0415] MS m / z (ESI): 439.1 [M+1].

[0416] The fourth step

[0417] (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

[0418] Add 1b (22 mg, 42.38 μmol) to the reaction flask, add 3 mL of N,N-dimethylformamide, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add triethylamine (4.3 mg, 42.49 μmol), add the crude 14c (23 mg, 51.1 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol), and stir the reaction in an ice bath for 40 minutes. Add 15 mL of water, extract with ethyl acetate (8 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (15 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by thin-layer chromatography with eluent system B to obtain the title product 14d (29 mg, yield: 79.9%).

[0419] MS m / z (ESI): 856.1 [M+1].

[0420] The fifth step

[0421] 2-((2-Aminoacetamido)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

[0422] Dissolve 14d (29 mg, 33.9 μmol) in 0.8 mL of dichloromethane, add 0.4 mL of diethylamine, and stir at room temperature for 1.5 hours. Concentrate the reaction solution under reduced pressure, add 1 mL of toluene and concentrate under reduced pressure again, repeating twice. Add 3 mL of n-hexane to the residue for slurrying, let it stand and pour out the supernatant, repeating three times. Concentrate the residue under reduced pressure and dry it with an oil pump to obtain the crude title product 14e (22 mg), and the product is directly used for the next reaction without purification.

[0423] MS m / z (ESI): 634.1 [M+1].

[0424] The sixth step

[0425] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A

[0426] 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-tetraazacyclohexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0427] The crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0 - 5 °C in an ice-water bath. 8 g (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 successively. The ice bath was removed and the mixture was stirred at room temperature for 1 hour to form compound 14. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic 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 title product (2 mg, 2 mg) was obtained.

[0428] MS m / z (ESI): 1088.4 [M+1].

[0429] Compound with a single configuration (shorter retention time):

[0430] UPLC analysis: retention time 1.18 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0431] Compound with a single configuration (longer retention time):

[0432] UPLC analysis: retention time 1.23 minutes, purity: 96% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0433] Example 1 - 15

[0434] 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-pentaazadocosan-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0435]

[0436] The first step

[0437] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylate 15b

[0438] 8b (500 mg, 1.35 mmol) was added to a reaction flask, and 6 mL of tetrahydrofuran was added. Benzyl 1-(hydroxymethyl)cyclopropane-1-carboxylate 15a (233 mg, 1.13 mmol; prepared by the method disclosed in Example 22-2 on page 262 of the patent application "EP2862856A1") was added to the flask. The flask was purged with argon three times, cooled to 0 - 5 °C in an ice-water bath, and sodium hydride (54 mg, 1.35 mmol) was added. The ice bath was removed, and the mixture was stirred at room temperature for 40 minutes. It was cooled to zero degree and 20 mL of ice water was added. The mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The combined organic phases were 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 the eluent system B to obtain the title product 15b (15 mg, yield: 2.5%).

[0439] MS m / z (ESI): 515.2 [M + 1].

[0440] The second step

[0441] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

[0442] 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, and palladium on carbon (3 mg, 10% content, dry type) was added. The flask was purged with hydrogen three times and stirred at room temperature for 4.5 hours. The reaction mixture was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to obtain the title product 15c (11 mg, yield: 89%).

[0443] MS m / z (ESI): 425.2 [M + 1].

[0444] The third step

[0445] (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

[0446] 1b (10 mg, 0.021 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, one drop of triethylamine was added, 15c (11 mg, 0.026 mmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol) was added, and after addition, the mixture was stirred at room temperature for 60 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing agent system B to obtain the title product 15d (19 mg, yield 87.0%).

[0447] MS m / z (ESI): 842.2 [M+1].

[0448] The fourth step

[0449] 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)cyclopropane-1-carboxamide 15e

[0450] 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 solution was concentrated under reduced pressure at 0 °C, 1 mL of toluene was added and concentrated under reduced pressure, and this was repeated twice; 3 mL of n-hexane was added for slurrying, the upper layer of n-hexane was decanted, and this was repeated three times; concentration under reduced pressure gave the crude title product 15e (13.9 mg), and the product was used directly in the next step without purification.

[0451] MS m / z (ESI): 620.1 [M+1].

[0452] The fifth step

[0453] 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-pentaazadocosyl)-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

[0454] Dissolve the crude product 15e (13.9 mg, 22.4 μmol) in 1 mL of N,N-dimethylformamide. Replace the gas with argon three times, cool it to 0 - 5 °C in an ice-water bath, add 8 g (15.8 mg, 33.4 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol), and stir the reaction at room temperature for 60 minutes. Purify the reaction solution by high-performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A - water (10 mmol NH4OAc): B - acetonitrile, gradient elution, flow rate: 18 mL / min), collect the corresponding fractions, and concentrate under reduced pressure to obtain the title product 15 (2.5 mg, yield: 10.3%).

[0455] MS m / z (ESI): 1074.2 [M+1].

[0456] 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), 2.41 - 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).

[0457] Examples 1 - 16

[0458] 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-pentaazadocosan-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16

[0459]

[0460]

[0461] The first step

[0462] 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

[0463] Ethyl 1-(hydroxymethyl)cyclobutane carboxylate 16a (250 mg, 1.58 mmol, supplier Alfa) was dissolved in methanol (2 mL) and water (1 mL), sodium hydroxide (126 mg, 3.15 mmol) was added, the temperature was raised to 40 °C, and the reaction was stirred for 3 hours. After cooling to room temperature, the organic solvent was removed by concentration under reduced pressure, back-extracted with diethyl ether (10 mL), and the aqueous phase was collected. The aqueous phase was adjusted to pH 3 - 4 with 6N hydrochloric acid aqueous solution, and concentrated under reduced pressure to obtain a solid. 3 mL of toluene was added, and the mixture was concentrated and dried by rotary evaporation under reduced pressure three times. Dried with an oil pump to obtain the crude title product 16b (206 mg), and the product was directly used in the next reaction without purification.

[0464] MS m / z (ESI, NEG): 129.2 [M - 1].

[0465] The second step

[0466] Benzyl 1-(hydroxymethyl)cyclobutane-1-carboxylate 16c

[0467] The crude product 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), anhydrous potassium carbonate (1.09 g, 7.90 mmol) and tetrabutylammonium iodide (29 mg, 78.51 μmol) were added, benzyl bromide (216 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature overnight. Filtered, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with the eluent system C to obtain the title product 16c (112 mg, yield: 32.1%).

[0468] MS m / z (ESI): 221.1 [M + 1].

[0469] The third step

[0470] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

[0471] 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to a reaction flask, 3 mL of tetrahydrofuran was added, and the mixture was purged with argon three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, and the mixture was stirred in an ice bath for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL) and chloroform (5 mL × 5). 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, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (71 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 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 with eluent system C to obtain the title product 16d (24 mg, yield: 16.7%).

[0472] MS m / z (ESI): 551.3 [M + 23].

[0473] The fourth step

[0474] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid 16e

[0475] 16d (12 mg, 22.7 μmol) was dissolved in a mixed solvent of 1.5 mL of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium on carbon (5 mg, 10% content) was added, and the mixture was purged with hydrogen three times. The mixture was stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain the crude title product 16e (10 mg), which was used directly in the next step without purification.

[0476] The fifth step

[0477] (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

[0478] 1b (7.5 mg, 0.014 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon 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 crude product 16e (10 mg) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6 mg, 0.026 mmol) was added, and the reaction was stirred in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using eluent system B to obtain the title product 16f (10.6 mg, yield 87.8%).

[0479] MS m / z (ESI): 856.2 [M+1].

[0480] The sixth step

[0481] 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

[0482] 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 solution was concentrated under reduced pressure, 2 mL of toluene was added and concentrated under reduced pressure, and this was repeated twice; 3 mL of n-hexane was added for trituration, the upper layer of n-hexane was poured out, and this was repeated three times. The residue was concentrated under reduced pressure to obtain the crude title product 16g (8 mg), and the product was used directly in the next step without purification.

[0483] MS m / z (ESI): 634.1 [M+1].

[0484] The seventh step

[0485] 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-pentaazadocosan-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16

[0486] Dissolve 16 g (8 mg) of the crude product in 1 mL of N,N-dimethylformamide, add 8 g (8.8 mg, 18.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (5.2 mg, 18.8 μmol), and stir the reaction at room temperature for 30 minutes. Purify the reaction solution by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A - water (10 mmol NH4OAc): B - acetonitrile, gradient elution, flow rate: 18 mL / min) to obtain the title product 16 (1.0 mg, yield: 7.2%).

[0487] MS m / z (ESI): 1088.0 [M+1].

[0488] Example 1 - 17

[0489] (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)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaaza-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0490]

[0491]

[0492] The first step

[0493] tert-Butyl 1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxatriacontane-31-carboxylate 17b

[0494] 1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacos-28-ol 17a (0.34 g, 0.67 mmol, supplier Bide) was dissolved in 10 mL of dichloromethane. 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 successively, and the mixture was stirred at room temperature for 3 hours. After filtration, the filtrate was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography with eluent system B to obtain the title product 17b (0.42 g, yield: 100%).

[0495] MS m / z (ESI): 636.3 [M+18].

[0496] The second step

[0497] tert-Butyl 29-hydroxy-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1-oate 17c

[0498] 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, palladium on carbon (110 mg, 10% content, dry type) was added, and the mixture was purged with hydrogen three times. Then it was stirred at 60 °C for 3 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to obtain the crude title product 17c (357 mg). The product was directly used for the next reaction without purification.

[0499] MS m / z (ESI): 546.2 [M+18].

[0500] The third step

[0501] tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1-oate 17d

[0502] 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, diphenylphosphoryl azide (279 mg, 1.014 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (206 mg, 1.353 mmol) were added, and the mixture was purged with argon three times. Then it was stirred at room temperature for 2 hours and then at 105 °C for 19 hours. The reaction solution was cooled to room temperature, concentrated, 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography with eluent system B to obtain the crude title product 17d (412 mg).

[0503] MS m / z (ESI): 571.3 [M+18].

[0504] The fourth step

[0505] tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacos-1-oate 17e

[0506] Dissolve 17d (230 mg, 0.415 mmol) in 8 mL of tetrahydrofuran, add palladium on carbon (58 mg, 10% content, dry type), displace with hydrogen three times, and stir the reaction at room temperature for 2 hours. Filter the reaction solution through diatomaceous earth, wash the filter cake with tetrahydrofuran, concentrate the filtrate to obtain the crude title product 17e (220 mg), and directly proceed to the next step without purifying the product.

[0507] MS m / z (ESI): 528.2 [M+1].

[0508] The fifth step

[0509] 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-azatriacont-31-oate 17f

[0510] Dissolve (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) in 10 mL of dichloromethane, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol), displace with argon three times, add the crude 17e (220 mg, 0.417 mmol), and stir the reaction at room temperature for 1 hour. Add 15 mL of water, extract with dichloromethane (8 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (15 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by silica gel column chromatography using eluent system B to obtain the title product 17f (122 mg, yield: 39.2%).

[0511] MS m / z (ESI): 747.2 [M+1].

[0512] The sixth step

[0513] 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-azatriacont-31-oic acid 17g

[0514] Dissolve 17f (122 mg, 0.163 mmol) in 0.8 mL of dichloromethane, add 0.4 mL of trifluoroacetic acid, and stir the reaction at room temperature for 1 hour. Dilute with 15 mL of dichloromethane and concentrate under reduced pressure; add 10 mL of n-hexane and concentrate under reduced pressure, repeat twice; then add 10 mL of toluene and concentrate under reduced pressure; slurry three times with a mixed solvent of 10 mL of n-hexane:ethyl ether = 5:1 until the pH is close to 7, concentrate, and dry under the oil pump to obtain the title product 17g (98 mg, yield: 86.8%).

[0515] MS m / z (ESI): 691.2 [M+1].

[0516] The seventh step

[0517] 2,4-Dimethoxybenzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropyl-1-carboxylate 17h

[0518] Dissolve 8d (164 mg, 0.40 mmol) in dichloromethane (5 mL), successively add 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.60 mmol) and 4-dimethylaminopyridine (5 mg, 0.041 mmol). After addition, stir the reaction at room temperature for 1 hour. Add 20 mL of water, shake and separate the layers. The aqueous phase is extracted with dichloromethane (8 mL×3), and the organic phases are combined. The organic phase is washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. Purify the obtained residue by silica gel column chromatography with the eluent system C to obtain the title product 17h (124 mg, yield: 55.4%).

[0519] MS m / z (ESI): 583.1 [M+23].

[0520] The eighth step

[0521] 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-pentaazaoctadec-17-yl)oxy)cyclopropyl-1-carboxylate 17j

[0522] Dissolve 17h (39 mg, 69.6 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 1 hour. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure again, repeat twice; add 3 mL of n-hexane for pulping, pour out the upper layer of n-hexane, repeat three times, and concentrate under reduced pressure. Dissolve the obtained crude product in 2 mL of N,N-dimethylformamide, add (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared by the method disclosed in Example 7-12 on page 13 of the specification of patent application "CN108853514A"), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 μmol), and stir at room temperature for 1 hour. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by thin-layer chromatography with eluent system B to obtain the title product 17j (48 mg, yield: 83.9%).

[0523] MS m / z (ESI): 822.0 [M+1].

[0524] The ninth step

[0525] (S)-1-((11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazapentadec-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

[0526] Dissolve 17j (48 mg, 58.4 μmol) in 1.4 mL of a dichloromethane solution of 3% (v / v) dichloroacetic acid, cool to 0-5 °C in an ice-water bath, add triethylsilane (21 mg, 180.6 μmol), and stir the reaction in an ice bath for 3 hours. Concentrate under reduced pressure to remove half of the organic solvent in the ice bath, add 5 mL of ether, let it rise to room temperature naturally for pulping, precipitate a white solid, filter, collect the filter cake, and dry it with an oil pump to obtain the title product 17k (33 mg, yield: 84.1%).

[0527] The tenth step

[0528] (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)cyclopropoxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridec-13-yl)carbamate 17l

[0529] Add 1b (20 mg, 42.4 μmol) to a reaction flask, add 1 mL of a 10% (v / v) methanol dichloromethane solution, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add a drop of triethylamine, and stir until 1b dissolves. Dissolve 17k (33 mg, 49.1 μmol) in 1 mL of a 10% (v / v) methanol dichloromethane solution, then add dropwise to the above reaction solution, and then add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol). Raise the temperature to room temperature and stir the reaction for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, let it stand for phase separation, and collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by thin-layer chromatography with developing agent system B to obtain the title product 17l (37 mg, yield: 80.2%).

[0530] MS m / z (ESI): 1090.1 [M + 1].

[0531] The eleventh step

[0532] (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)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatetratriacontan-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0533] Dissolve 17l (15.5 mg, 14.23 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 1.5 hours. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure again, repeat twice; add 3 mL of n-hexane for pulping, pour out the upper layer of n-hexane, repeat three times. Concentrate under reduced pressure, and then dry with an oil pump. Dissolve the obtained crude product in 1 mL of N,N-dimethylformamide, add 17 g (11 mg, 15.92 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 μmol), displace with argon three times, and stir at room temperature for 30 minutes. Purify the reaction solution by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A - water (10 mmol NH4OAc): B - acetonitrile, gradient elution, flow rate: 18 mL / min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product 17 (6 mg, yield: 27.4%).

[0534] MS m / z (ESI): 1556.4 [M + 18].

[0535] 1 1H NMR (400 MHz, DMSO-d6): δ8.98 (d, 1H), 8.76 (s, 1H), 8.20 (br, 1H), 8.12 - 7.95 (m, 3H), 7.93 - 7.76 (m, 2H), 7.75 - 7.66 (m, 2H), 7.24 (s, 1H), 7.20 - 7.05 (m, 6H), 6.97 (s, 1H), 6.64 (br, 1H), 6.55 (d, 1H), 6.47 (s, 1H), 5.61 - 5.52 (m, 2H), 5.37 (s, 1H), 5.33 - 5.23 (m, 2H), 5.18 (s, 1H), 5.13 (s, 1H), 5.05 (s, 1H), 5.00 (s, 1H), 4.65 - 4.55 (m, 2H), 4.53 - 4.45 (m, 1H), 4.38 - 4.28 (m, 2H), 3.84 (s, 2H), 3.67 (d, 3H), 3.60 - 3.40 (m, 33H), 3.18 (d, 1H), 3.15 - 3.08 (m, 3H), 2.28 (s, 3H), 2.00 - 1.92 (m, 3H), 1.85 (s, 2H), 1.82 - 1.73 (m, 2H), 1.68 - 1.52 (m, 4H), 1.29 - 1.15 (m, 3H), 0.86 - 0.76 (m, 5H).

[0536] Example 1 - 18

[0537] (1r,4r)-N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0538]

[0539]

[0540] The first step

[0541] (R)-Benzyl 2-cyclopropyl-2-hydroxyacetate 18a

[0542] (S)-Benzyl 2-cyclopropyl-2-hydroxyacetate 18b

[0543] Dissolve 2a (7.4 g, 63.7 mmol) in 200 mL of acetonitrile, and successively add potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide (500 mg, 1.36 mmol). Stir the reaction mixture at room temperature for 16 hours, filter it through diatomaceous earth, wash the filter cake with ethyl acetate (10 mL), combine the filtrates and concentrate them under reduced pressure. Purify the resulting residue (4.1 g) by silica gel column chromatography with eluent system C, and further purify it by chiral resolution to obtain the title product 18a (1.1 g) and 18b (1.2 g).

[0544] The second step

[0545] (R)-Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazoundec-11-oate 18c

[0546] 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 for 10 minutes in the ice - water bath. Ethyl acetate (30 mL) and water (20 mL) were added, and the mixture was allowed to stand for layer 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 hours. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). 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 with eluent system C to obtain the title product 18c (1.3 g, yield: 30.0%).

[0547] MS m / z (ESI): 515.2 [M + 1].

[0548] The third step

[0549] (R)-10 - Cyclopropyl - 1-(9H - fluoren - 9 - yl)-3,6 - dioxo - 2,9 - dioxo - 4,7 - diazaundec - 11 - oic acid 18d

[0550] 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), palladium - carbon (260 mg, 10% content, dry type) was added, and the mixture was purged with hydrogen three times and stirred at room temperature for 5 hours. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to obtain the crude title product 18d (980 mg), and the product was directly used in the next step without purification.

[0551] MS m / z (ESI): 425.1 [M + 1].

[0552] The fourth step

[0553] (R)-10 - Cyclopropyl - 1-(9H - fluoren - 9 - yl)-3,6 - dioxo - 2,9 - dioxo - 4,7 - diazaundec - 11 - yl 2,4 - dimethoxybenzyl ester 18e

[0554] The crude product 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 mL), and 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (664 mg, 3.46 mmol) and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added. The mixture was stirred at room temperature for one hour. The organic solvent was removed by concentration under reduced pressure. 20 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined. The combined organic phase was washed with saturated sodium chloride solution (30 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by column chromatography with eluent system C to give the title product 18e (810 mg, yield: 61.1%).

[0555] MS m / z(ESI): 575.0[M+1].

[0556] The fifth step

[0557] 2,4-Dimethoxybenzyl (R)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetate 18f

[0558] 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and concentrated under reduced pressure again, and this was repeated twice; 3 mL of n-hexane was added for trituration, the upper n-hexane layer was decanted, and this was repeated three times. The residue was concentrated under reduced pressure to give the crude title product 18f (21 mg), and the product was used directly in the next step without purification.

[0559] The sixth step

[0560] 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-pentaazadocos-20-oate 18g

[0561] The crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, 17i (29 mg, 57.8 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined. The combined organic phase was washed with saturated sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by thin layer chromatography with eluent system B to give the title product 18g (37 mg, yield: 77.1%).

[0562] MS m / z (ESI): 853.0 [M+18].

[0563] The seventh step

[0564] (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-pentaazaeicos-20-oic acid 18h

[0565] Dissolve 18 g (37 mg, 44.3 μmol) in 1.4 mL of a dichloromethane solution of 3% (v / v) dichloroacetic acid, cool it to 0 - 5 °C in an ice-water bath, add triethylsilane (15.4 mg, 132.4 μmol), and stir the reaction in an ice bath for 3 hours. Concentrate under reduced pressure in an ice bath to remove half of the organic solvent, add 5 mL of diethyl ether, let it rise to room temperature and stir to form a slurry, precipitate a white solid, filter, collect the filter cake, and dry it with an oil pump to obtain the title product 18h (24 mg, yield: 79.1%).

[0566] MS m / z (ESI): 708.2 [M+23].

[0567] The eighth step

[0568] (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-tetraazahexadec-16-yl)carbamate 18i

[0569] Add 1b (30 mg, 63.6 μmol) to a reaction flask, add 1 mL of a 10% (v / v) methanol in dichloromethane solution, displace with argon three times, cool to 0 - 5 °C in an ice - water bath, add a drop of triethylamine, and stir until 1b dissolves. Dissolve 18h (65 mg, 94.8 μmol) in 1 mL of a 10% (v / v) methanol in dichloromethane solution, then dropwise add it to the above reaction solution, and then add 4-(4,6 - dimethoxy - 1,3,5 - triazin - 2 - yl)-4 - methylmorpholinium chloride (27 mg, 97.6 μmol). Raise the temperature to room temperature and stir the reaction for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, let it stand for phase separation, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by thin - layer chromatography with eluent system B to obtain the title product 18i (25 mg, yield: 35.6%).

[0570] MS m / z(ESI): 1104.4[M + 1].

[0571] The ninth step

[0572] (S)-2-(2-(2 - Aminoacetamido)acetamido)-N-(2-((((R)-1 - cyclopropyl - 2-(((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benz[de]pyrano[3',4':6,7]indolizino[1,2 - b]quinolin - 1 - yl)amino)-2 - oxoethoxy)methyl)amino)-2 - oxoethoxy)-3 - phenylpropanamide 18j

[0573] Dissolve 18i (12 mg, 10.9 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 1.5 hours. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure twice, add 3 mL of n - hexane for pulping, pour out the upper layer of n - hexane three times, and concentrate under reduced pressure to obtain the crude title product 18j (10 mg), and the product is directly used for the next reaction without purification.

[0574] MS m / z(ESI): 881.0[M + 1].

[0575] The tenth step

[0576] (1r,4r)-N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0577] Dissolve the crude product 18j (10 mg) in 1 mL of N,N-dimethylformamide, add 17 g (8.5 mg, 12.3 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol), and stir at room temperature for 30 minutes. Filter the reaction solution and purify it 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), collect the corresponding fractions, concentrate under reduced pressure to obtain the title product 18 (9.5 mg, yield: 56.2%).

[0578] MS m / z (ESI): 1570.2 [M+18].

[0579] 11H 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).

[0580] Examples 1 - 19

[0581] (1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetracont-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0582]

[0583]

[0584] The first step

[0585] (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-yl benzoate 19a

[0586] 18b (252 mg, 1.22 mmol) was added to a reaction flask, 4 mL of dichloromethane was added, and the mixture was purged with argon three times. The temperature was lowered to 0 - 5 °C in an ice - water bath, and lithium tert - butoxide (98 mg, 1.22 mmol) was added. The reaction was stirred for 15 minutes in the ice - water bath until it became clear. Then 8b (300 mg, 814.3 μmol) was added, and the mixture was stirred for 2.5 hours in the ice - water bath. Water (10 mL) was added, and the layers were separated. The aqueous phase was extracted with dichloromethane (8 mL×2). The combined organic phases were washed with water (10 mL×1) and saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography with eluent system C to give the title product 19a (282 mg, yield: 67.2%).

[0587] The second step

[0588] (S)-10 - Cyclopropyl - 1-(9H - fluoren - 9 - yl)-3,6 - dioxo - 2,9 - dioxo - 4,7 - diazaundec - 11 - oic acid 19b

[0589] 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, palladium - carbon (84 mg, 10% content, dry type) was added, and the mixture was purged with hydrogen three times. The reaction was stirred at room temperature for 3 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated to obtain the crude product of the title product 19b (230 mg). The product was directly used for the next step without purification.

[0590] The third step

[0591] 2,4 - Dimethoxybenzyl (S)-10 - cyclopropyl - 1-(9H - fluoren - 9 - yl)-3,6 - dioxo - 2,9 - dioxo - 4,7 - diazaundec - 11 - oate 19c

[0592] The crude 19b (230 mg, 541.8 μmol) was dissolved in 7 mL of dichloromethane. 2,4 - Dimethoxybenzyl alcohol (136.7 mg, 812.7 μmol), 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide hydrochloride (155 mg, 808.5 μmol), and 4 - dimethylaminopyridine (6.6 mg, 53.5 μmol) were added successively. The reaction was stirred at room temperature for 16 hours. The reaction solution was diluted with 10 mL of dichloromethane, washed with water (10 mL×1) and saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified by thin - layer chromatography with eluent system B to give the title product 19c (159 mg, yield: 51.0%)

[0593] The fourth step

[0594] 2,4-Dimethoxybenzyl (S)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetate 19d

[0595] Dissolve 19c (60 mg, 104.4 μmol) in 1 mL of dichloromethane, add 0.5 mL of diethylamine, and stir at room temperature for 1 hour. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure again, repeat twice; add 3 mL of n-hexane for slurrying, pour out the upper layer of n-hexane, repeat three times, and concentrate under reduced pressure to obtain the crude title product 19d (21 mg). The product is directly used for the next reaction without purification.

[0596] The fifth step

[0597] 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-pentaazaeicos-20-oate 19e

[0598] Dissolve the crude 19d (36 mg, 102.2 μmol) in 4 mL of N,N-dimethylformamide, add 17i (52 mg, 103.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 μmol), and stir at room temperature for 1 hour. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by thin layer chromatography using the eluent system B to obtain the title product 19e (70 mg, yield: 80.2%).

[0599] The sixth step

[0600] (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-pentaazaeicos-20-oic acid 19f

[0601] Dissolve 19e (70 mg, 83.7 μmol) in 2.5 mL of a 3% (v / v) solution of dichloroacetic acid in dichloromethane, cool to 0 - 5 °C in an ice-water bath, add triethylsilane (29 mg, 249.4 μmol), and stir the reaction in an ice bath for 3 hours. Concentrate under reduced pressure in an ice bath to remove half of the organic solvent, add 5 mL of ether, let it rise to room temperature naturally for slurrying, precipitate a white solid, filter, collect the filter cake, and dry it with an oil pump to obtain the title product 19f (57 mg, yield: 99.2%).

[0602] The seventh step

[0603] (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-tetraazahexadec-16-yl)carbamate 19g

[0604] Add 1b (30 mg, 63.6 μmol) to a reaction flask, add 1 mL of a 10% (v / v) methanol in dichloromethane solution, displace with argon three times, cool to 0 - 5 °C in an ice-water bath, add a drop of triethylamine, and stir until 1b dissolves. Dissolve 19f (57 mg, 83.1 μmol) in 1 mL of a 10% (v / v) methanol in dichloromethane solution, then dropwise add it to the above reaction solution, and then add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol). Raise the temperature to room temperature and stir the reaction for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, let it stand for liquid separation, and collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by thin-layer chromatography using eluent system B to obtain the title product 19g (56 mg, yield: 79.8%).

[0605] MS m / z (ESI): 1103.1 [M+1].

[0606] The Eighth Step

[0607] (S)-2-(2-(2-Aminoacetamido)acetamido)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,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

[0608] Dissolve 19 g (4.6 mg, 4.16 μmol) in 1.5 mL of dichloromethane, add 0.75 mL of diethylamine, and stir at room temperature for 1.6 hours. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure again, repeat twice, add 3 mL of n-hexane for pulping, pour out the upper layer of n-hexane, repeat three times, and concentrate under reduced pressure to obtain the crude product of the title compound 19h (4.0 mg). The product is directly used in the next reaction without purification.

[0609] The ninth step

[0610] (1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0611] Dissolve the crude product 19h (4.0 mg) in 1 mL of N,N-dimethylformamide, add 17 g (2.9 mg, 4.2 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol), and stir at room temperature for 40 minutes. Filter the reaction solution and purify it by high performance liquid chromatography (separation conditions: chromatographic 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), collect the corresponding fractions, and concentrate under reduced pressure to obtain the title compound 19 (2.1 mg, yield: 32.4%).

[0612] 11H 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).

[0613] Examples 1 - 20 (Reference Examples)

[0614]

[0615] The title compound 20 was synthesized according to the method provided in Example 58 on page 163 of the specification of the reference patent "CN104755494A".

[0616] The following antibodies were prepared by conventional antibody methods. For example, after vector construction, eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) were transfected, and after expression and purification, the antibodies were obtained.

[0617] The following is the sequence of Trastuzumab:

[0618] Light chain

[0619]

[0620] Heavy chain

[0621]

[0622] The following is the sequence of Pertuzumab:

[0623] Light chain

[0624]

[0625] Heavy chain

[0626]

[0627] The following is the sequence of B7H3 antibody 1F9DS:

[0628] Light chain

[0629]

[0630] Heavy chain

[0631]

[0632] Examples 1 - 21 ADC-1

[0633]

[0634] 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 antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 2.5 mL, 9.96 mg / mL, 0.168 μmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken for further reaction.

[0635] Compound 10 - shorter retention time compound (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (5.0 mg / mL, 1.1 mL) of the exemplary product ADC-1 of the general formula FADC-1, which was stored at 4 °C.

[0636] The average value was calculated by UV-HPLC: n = 5.09.

[0637] Examples 1 - 22 ADC-2

[0638]

[0639] At 37 °C, an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 2.5 mL, 9.96 mg / mL, 0.168 μmol) was added with an aqueous solution of tris(2-carboxyethyl)phosphine prepared (10 mM, 0.082 mL, 0.82 μmol), placed in a water bath oscillator, and reacted with shaking at 37 °C for 3 hours to stop the reaction; the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken for further reaction.

[0640] Compound 10 - longer retention time compound (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL solution, placed in a water bath oscillator, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-2 of the general formula FADC-1 (4.95 mg / mL, 1.1 mL), and stored at 4 °C.

[0641] The average value was calculated by UV-HPLC: n = 7.39.

[0642] Example 1-23 ADC-3

[0643]

[0644] At 37 °C, an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 2.5 mL, 9.96 mg / mL, 0.168 μmol) was added with an aqueous solution of tris(2-carboxyethyl)phosphine prepared (10 mM, 0.082 mL, 0.82 μmol), placed in a water bath oscillator, and reacted with shaking at 37 °C for 3 hours to stop the reaction; the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken for further reaction.

[0645] Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL solution, placed in a water bath oscillator, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-3 of the general formula FADC-3 (5.24 mg / mL, 1.1 mL), and stored at 4 °C.

[0646] The average value calculated by UV-HPLC: n = 7.36.

[0647] Examples 1-24 ADC-4

[0648]

[0649] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) prepared was added to an aqueous PBS buffer solution of antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 3.74 mL, 13.38 mg / mL, 0.338 μmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C with a water bath, diluted to 6.7 mg / mL, and 1.3 mL of the solution was taken out for the subsequent reaction.

[0650] Compound 9 - shorter 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 of solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.72 mg / mL, 2.36 mL) of the exemplary product ADC-4 of the general formula FADC-4A, and stored at 4 °C.

[0651] The average value calculated by UV-HPLC: n = 7.39.

[0652] Examples 1-25 ADC-5

[0653]

[0654] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) prepared was added to an aqueous PBS buffer solution of antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 3.0 mL, 6.70 mg / mL, 0.136 μmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C with a water bath, and 0.614 mL of the solution was taken out for the subsequent reaction.

[0655] Dissolve compound 9 - shorter retention time compound 9 - A (0.5 mg, 0.42 μmol) in 0.031 mL of DMSO, add it to the above 0.614 mL solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution with a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (3.08 mg / mL, 0.82 mL) of the exemplary product ADC - 5 of the general formula FADC - 4A, and store it at 4 °C.

[0656] UV - HPLC calculated average value: n = 3.16.

[0657] Example 1 - 26 ADC - 6

[0658]

[0659] Under the condition of 37 °C, add the prepared aqueous solution of tris(2 - carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) to the PBS buffer aqueous solution of the antibody trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5; 3.74 mL, 13.38 mg / mL, 0.338 μmol), place it in a water bath oscillator, and react with shaking at 37 °C for 3 hours to stop the reaction; cool the reaction solution to 25 °C with a water bath, dilute it to 6.7 mg / mL, and take out 0.75 mL of the solution for further reaction.

[0660] Dissolve compound 9 - longer retention time compound 9 - B (0.68 mg, 0.63 μmol) in 0.10 mL of DMSO, add it to the above 0.75 mL solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution with a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (1.78 mg / mL, 1.78 mL) of the exemplary product ADC - 6 of the general formula FADC - 4B, and store it at 4 °C.

[0661] UV - HPLC calculated average value: n = 3.94.

[0662] Example 1 - 27 ADC - 7

[0663]

[0664] 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 at pH = 6.5; 5.0 mL, 10 mg / mL, 0.338 μmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 1.0 mL of the solution was taken for further reaction.

[0665] Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL of DMSO and added to the above 1.0 mL solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-7 of the general formula FADC-7 (1.42 mg / mL, 2.15 mL), which was stored at 4 °C.

[0666] The average value was calculated by UV-HPLC: n = 6.91.

[0667] Example 1-28 ADC-8

[0668]

[0669] 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 at pH = 6.5; 5.0 mL, 10 mg / mL, 0.338 μmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 1.6 mL of the solution was taken for further reaction.

[0670] Compound 10 - shorter retention time compound (1.04 mg, 1.0 μmol) was dissolved in 0.1 mL of DMSO and added to the above 1.6 mL solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-8 of the general formula FADC-8 (2.14 mg / mL, 2.31 mL), which was stored at 4 °C.

[0671] UV-HPLC calculated average value: n = 6.58.

[0672] Examples 1 - 29 ADC-9

[0673]

[0674] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) prepared was added to an aqueous PBS buffer solution of antibody pertuzumab (0.05 M PBS buffer solution with pH = 6.5; 5.0 mL, 10 mg / mL, 0.338 μmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 0.8 mL of the solution was taken for further reaction.

[0675] Compound 9 - shorter 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 of solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH = 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-9 in PBS buffer (2.27 mg / mL, 1.11 mL), and stored at 4 °C.

[0676] UV-HPLC calculated average value: n = 3.16.

[0677] Examples 1 - 30 ADC-10

[0678]

[0679] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) prepared was added to an aqueous PBS buffer solution of antibody trastuzumab (0.05 M PBS buffer solution with pH = 6.5; 10.0 mg / mL, 0.574 mL, 38.78 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0680] Compound 14 - shorter retention time compound (0.64 mg, 588 nmol) was dissolved in 40 μl of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (5.48 mg / mL, 1.03 mL) of the exemplary product ADC - 10 of the general formula FADC - 10, and stored at 4 °C.

[0681] UV - Vis calculated average value: n = 6.25.

[0682] Example 1 - 31 ADC - 11

[0683]

[0684] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) prepared was added to an aqueous PBS buffer solution of the antibody trastuzumab (pH = 6.5, 0.05 M PBS buffer aqueous solution; 10.0 mg / mL, 0.646 mL, 43.64 nmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C using a water bath.

[0685] Compound 14 - longer retention time compound (0.72 mg, 662 nmol) was dissolved in 40 μl of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (2.13 mg / mL, 1.87 mL) of the exemplary product ADC - 11 of the general formula FADC - 10, and stored at 4 °C.

[0686] UV - Vis calculated average value: n = 7.03.

[0687] Example 1 - 32 ADC - 12

[0688]

[0689] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol) prepared was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.726 mL, 49.05 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0690] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-12 of the general formula FADC-12 (3.34 mg / mL, 1.45 mL), which was stored at 4 °C.

[0691] UV-Vis calculated average value: n = 6.93.

[0692] Example 1-33 ADC-13

[0693]

[0694] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) prepared was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.287 mL, 19.39 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0695] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC-13 of the general formula FADC-13 (2.37 mg / mL, 0.88 mL), which was stored at 4 °C.

[0696] UV-Vis calculated average value: n = 6.53.

[0697] Examples 1 - 34 ADC - 14

[0698]

[0699] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution with pH = 6.5; 10.0 mg / mL, 0.592 mL, 40.0 nmol) was added with an aqueous solution of prepared tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol), placed in a water bath oscillator, and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0700] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μl of DMSO, added to the above - mentioned reaction solution, placed in a water bath oscillator, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution with pH = 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (0.30 mg / mL, 12.0 mL) of the exemplary product ADC - 14 of the FADC - 14 general formula, and stored at 4 °C.

[0701] UV - Vis calculated average value: n = 7.61.

[0702] Examples 1 - 35 ADC - 15

[0703]

[0704] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution with pH = 6.5; 10.0 mg / mL, 0.592 mL, 40.0 nmol) was added with an aqueous solution of prepared tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol), placed in a water bath oscillator, and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0705] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (0.32 mg / mL, 11.8 mL) of the exemplary product ADC-15 of the general formula FADC-15, which was stored at 4 °C.

[0706] UV-Vis calculated average value: n = 7.89.

[0707] Example 1-36 ADC-16

[0708]

[0709] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) prepared was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer aqueous solution with a pH of 6.5; 10.0 mg / mL, 0.53 mL, 35.8 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0710] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (0.32 mg / mL, 12.0 mL) of the exemplary product ADC-16 of the general formula FADC-16, which was stored at 4 °C.

[0711] UV-Vis calculated average value: n = 7.43.

[0712] Example 1-37 ADC-17

[0713]

[0714] At 37 °C, an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 2.0 mL, 135.12 nmol) was added to an aqueous solution of prepared tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0715] Compound 9 - shorter retention time compound 9 - A (2.22 mg, 2067 nmol) was dissolved in 175 μl of DMSO and added to the above reaction solution. It was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH = 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (1.32 mg / mL, 12.0 mL) of the exemplary product ADC - 17 of the general formula FADC - 4A, which was stored at 4 °C.

[0716] UV - Vis calculated average value: n = 5.42.

[0717] Example 1 - 38 ADC - 18 (reference example)

[0718]

[0719] At 37 °C, an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 1.5 mL, 101.3 nmol) was added to an aqueous solution of prepared tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0720] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. It was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH = 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (0.79 mg / mL, 13.0 mL) of the exemplary product ADC - 18 of the general formula FADC - 18, which was stored at 4 °C.

[0721] UV - Vis calculated average value: n = 7.23.

[0722] Examples 1 - 39 ADC - 19

[0723]

[0724] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at 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) prepared (10 mM, 46.9 μL, 469 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0725] Compound 9 - shorter retention time compound 9 - A (2.0 mg, 1862 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.73 mg / mL, 13.0 mL) of the exemplary product ADC - 19 of the general formula FADC - 4A, which was stored at 4 °C.

[0726] UV - Vis calculated average value: n = 6.26.

[0727] Examples 1 - 40 ADC - 20

[0728]

[0729] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at 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) prepared (10 mM, 51.7 μL, 517 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0730] Dissolve Compound 10 - longer retention time compound (2.0 mg, 1815 nmol) in 100 μl of DMSO, add it to the above reaction solution, place it in a water bath shaker, react with shaking at 25 °C for 3 hours, and stop the reaction. Desalt and purify the reaction solution with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (0.73 mg / mL, 13.0 mL) of the exemplary product ADC - 20 of the general formula FADC - 1, and store it at 4 °C.

[0731] UV - Vis calculated average value: n = 7.43.

[0732] Example 1 - 41 ADC - 21 (Reference Example)

[0733]

[0734] Under the condition of 37 °C, add the prepared aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) to the PBS buffer aqueous solution of the antibody trastuzumab (pH = 6.5, 0.05 M PBS buffer aqueous solution; 10.0 mg / mL, 1.86 mL, 125.4 nmol), place it in a water bath shaker, react with shaking at 37 °C for 3 hours, and stop the reaction. Cool the reaction solution to 25 °C with a water bath.

[0735] Dissolve Compound 20 (2.07 mg, 2001 nmol) in 150 μl of DMSO, add it to the above reaction solution, place it in a water bath shaker, react with shaking at 25 °C for 3 hours, and stop the reaction. Desalt and purify the reaction solution with a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (2.91 mg / mL, 4.44 mL) of the exemplary product ADC - 21 of the general formula FADC - 18, and store it at 4 °C.

[0736] UV - Vis calculated average value: n = 7.23.

[0737] Example 1 - 42 ADC - 22

[0738]

[0739] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 1.88 mL, 127.2 nmol) was added with an aqueous solution of prepared tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0740] Compound 9 - shorter retention time compound 9 - A (2.1 mg, 1955 nmol) was dissolved in 150 μl of DMSO and added to the above reaction solution. It was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (3.56 mg / mL, 3.98 mL) of the exemplary product ADC - 22 of the FADC - 4A general formula, and stored at 4 °C.

[0741] UV - Vis calculated average value: n = 6.79.

[0742] Example 1 - 43 ADC - 23 (Reference Example)

[0743]

[0744] At 37 °C, an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 345 mL, 23.31 μmol) was added with an aqueous solution of prepared tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3.5 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0745] Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified successively through an ultrafiltration membrane bag with PBS buffer aqueous solution containing 2% (v / v) MeCN and 1% (v / v) DMSO (0.05 M PBS buffer aqueous solution with pH = 6.5), succinic acid buffer aqueous solution (0.01 M succinic acid buffer aqueous solution with pH = 5.3), and then sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL. After bottling and freeze-drying, a lyophilized powder sample of the exemplary product ADC-23 of the FADC-18 general formula was obtained and stored at 4 °C.

[0746] UV-Vis calculated average value: n = 7.05.

[0747] Example 1 - 44 ADC-24

[0748]

[0749] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol) prepared was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 332 mL, 22.43 μmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3.5 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C with a water bath.

[0750] Compound 9 - the shorter 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 above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified successively through an ultrafiltration membrane bag with PBS buffer aqueous solution containing 4% (v / v) MeCN and 2% (v / v) DMSO (0.05 M PBS buffer aqueous solution with pH = 6.5), succinic acid buffer aqueous solution (0.01 M succinic acid buffer aqueous solution with pH = 5.3), and then sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL. After bottling and freeze-drying, a lyophilized powder sample of the exemplary product ADC-24 of the FADC-4A general formula was obtained and stored at 4 °C.

[0751] UV-Vis calculated average value: n = 7.07.

[0752] Example 1 - 45 ADC-25

[0753]

[0754] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol) prepared was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 2.14 mL, 144.60 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0755] Compound 9 - shorter retention time compound 9 - A (3.0 mg, 2793 nmol) was dissolved in 150 μl of DMSO and added to the above reaction solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC - 25 of the FADC - 25 general formula (1.28 mg / mL, 13.0 mL), and stored at 4 °C.

[0756] UV - Vis calculated average value: n = 6.87.

[0757] Example 1 - 46 ADC - 26 (reference example)

[0758]

[0759] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) prepared was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.89 mL, 60.14 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0760] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution of the exemplary product ADC - 26 of the FADC - 26 general formula (1.61 mg / mL, 4.0 mL), and stored at 4 °C.

[0761] UV-Vis calculated average value: n = 6.15.

[0762] Examples 1 - 47 ADC-27

[0763]

[0764] At 37 °C, to the PBS buffer aqueous solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 0.89 mL, 60.14 nmol) was added the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0765] Compound 9 - shorter retention time compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 μl DMSO and added to the above reaction solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of the exemplary product ADC-27 of the FADC-25 general formula (1.94 mg / mL, 3.5 mL), which was stored at 4 °C.

[0766] UV-Vis calculated average value: n = 6.11.

[0767] Examples 1 - 48 ADC-28 (reference example)

[0768]

[0769] At 37 °C, to the PBS buffer aqueous solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 2.36 mL, 159.47 nmol) was added the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol). It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0770] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (1.29 mg / mL, 13.0 mL) of the exemplary product ADC-28 of the general formula FADC-26, which was stored at 4 °C.

[0771] UV-Vis calculated average value: n = 7.46.

[0772] Examples 1-49 ADC-29

[0773]

[0774] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol) prepared was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with a pH of 6.5; 10.0 mg / mL, 0.80 mL, 50.06 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0775] Compound 9 - shorter retention time compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (2.63 mg / mL, 2.4 mL) of the exemplary product ADC-29 of the general formula FADC-25, which was stored at 4 °C.

[0776] UV-Vis calculated average value: n = 7.24.

[0777] Examples 1-50 ADC-30 (reference example)

[0778]

[0779] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) prepared was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.86 mL, 58.4 nmol). The mixture was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0780] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.61 mg / mL, 4.0 mL) of the exemplary product ADC-30 of the general formula FADC-26, which was stored at 4 °C.

[0781] UV-Vis calculated average value: n = 6.15.

[0782] Example 1-51 ADC-31

[0783]

[0784] At 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) prepared was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.89 mL, 60.14 nmol). The mixture was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0785] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.47 mg / mL, 4.5 mL) of the exemplary product ADC-31 of the general formula FADC-31, which was stored at 4 °C.

[0786] UV-Vis calculated average value: n = 6.33.

[0787] Analysis of drug loading of ADC stock solution

[0788] Experimental Purpose and Principle

[0789] The ADC stock solution is a drug of the antibody conjugate type. The mechanism of treating diseases depends on the targeting of the antibody to transport the toxin molecule into the cell and then kill the cell. The drug loading plays a decisive role in the drug efficacy. The ultraviolet method was used to determine the drug loading of the ADC stock solution.

[0790] Experimental Method

[0791] After placing the cuvette filled with sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, and subtracting the solvent blank, then place the cuvette filled with the test solution in the sample measurement absorption cell, and measure the absorbance at 280 nm and 370 nm.

[0792] Result Calculation: The ultraviolet spectrophotometry (instrument used: Thermo nanodrop2000 ultraviolet spectrophotometer) was used to determine the drug loading of the ADC stock solution. 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 this wavelength, that is:

[0793] (1) A 280nm = ε mab-280 bC mab + ε Drug-280 bC Drug

[0794] ε Drug-280 : The average molar extinction coefficient of the drug at 280 nm is 5100;

[0795] C Drug : The concentration of the drug;

[0796] ε mab-280 : The average molar extinction coefficient of trastuzumab stock solution or pertuzumab stock solution at 280 nm is 214600;

[0797] C mab : The concentration of trastuzumab stock solution or pertuzumab stock solution;

[0798] b: The optical path length is 1 cm.

[0799] Similarly, the total absorbance value equation of the sample at 370 nm can be obtained:

[0800] (2) A 370nm = ε mab-370 bC mab + ε Drug-370 bC Drug

[0801] ε Drug-370:The average molar extinction coefficient of the drug at 370 nm is 19,000;

[0802] C Drug :The concentration of the drug;

[0803] ε mab-370 :The extinction coefficient of the trastuzumab or pertuzumab stock solution at 370 nm is 0;

[0804] C mab :The concentration of the trastuzumab stock solution;

[0805] b: The optical path length is 1 cm.

[0806] Combining equations (1) and (2) with the extinction coefficient and concentration data of the monoclonal antibody and the drug at two detection wavelengths, the drug loading can be calculated.

[0807] Drug loading = C Drug / C mab .

[0808] Biological evaluation

[0809] Test Example 1-1: In vitro proliferation inhibition test of the compounds disclosed in this disclosure against tumor cells

[0810] I. Test purpose

[0811] The purpose of this experiment is to detect the inhibitory activity of the drug compounds disclosed in this disclosure against the in vitro proliferation of U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). Cells were treated in vitro with different concentrations of the compound. After 6 days of culture, the CTG ( Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent was used to detect the proliferation of the cells, and the in vitro activity of the compound was evaluated based on the IC50 value.

[0812] II. Experimental method

[0813] Taking the test method for the in vitro proliferation inhibition of U87MG cells as an example, it is used to illustrate the method for testing the in vitro proliferation inhibition activity of the compounds disclosed in this disclosure against tumor cells. This method is also applicable to, but not limited to, the in vitro proliferation inhibition activity test of other tumor cells.

[0814] 1. Cell culture: U87MG and SK-BR-3 cells were cultured with EMEM medium containing 10% FBS (GE, catalog number SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, catalog number 16600-108), respectively.

[0815] 2. Cell preparation: Take U87MG and SK-BR-3 cells in the logarithmic growth phase, wash once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3mL trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2-3min. After the cells are completely digested, add 10-15mL cell culture medium to wash the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, and then add 10-20mL cell culture medium to resuspend the cells to make a single cell suspension.

[0816] 3. Cell plating: Mix the single cell suspension of U87MG and SK-BR-3, and adjust the live cell density to 2.75×10 3 cells / mL and 8.25×10 3 cells / mL, mix the cell suspension after density adjustment, and add 180μL / well to a 96-well cell culture plate. Add only 200μL of culture medium to the peripheral wells of the 96-well plate. Incubate the culture plate in an incubator for 24 hours (37°C, 5% CO2).

[0817] 4. Compound preparation: Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) and prepare a storage solution with an initial concentration of 10 mM.

[0818] The initial concentration of the small molecule compound was 500 nM, and the preparation method was as follows.

[0819] Add 30 μL of different samples to be tested to the first column of a 96-well U-bottomed drug plate, with a sample concentration of 100 μM; add 20 μL of DMSO to each well in columns 2 to 11. Take 10 μL of the sample in the first column and add it to 20 μL of DMSO in the second column, mix well, take 10 μL to the third column, and so on to column 10. Take 5 μL of the drug in each well of the drug plate and add it to 95 μL of EMEM culture medium, mix well, and set aside.

[0820] The starting concentration of ADC was 10 nM or 500 nM, and the preparation method was as follows.

[0821] Add 100 μL of different samples to be tested into the first column of the 96-well plate, with the sample concentrations being 100 nM or 5 μM; add 100 μL of PBS to each well in the second to eleventh columns. Take 50 μL of the samples in the first column and add them to 100 μL of PBS in the second column, mix well, then take 50 μL and add it to the third column, and so on for three-fold dilution to the tenth column.

[0822] 5. Sample addition operation: Add 20 μL of the prepared samples with different concentrations to be tested into the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37 °C, 5% CO2).

[0823] 6. Color development operation: Take out the 96-well cell culture plate and add 90 μL of CTG solution to each well, then incubate at room temperature for 10 minutes.

[0824] 7. Plate reading operation: Take out the 96-well cell culture plate and place it in an enzyme-linked immunosorbent assay instrument (BMG labtech, PHERAstar FS), and use the enzyme-linked immunosorbent assay instrument to measure chemiluminescence.

[0825] III. Data analysis

[0826] Process and analyze the data using Microsoft Excel and Graphpad Prism 5. See Table 1 below for the experimental results.

[0827] Table 1. IC of the small molecule fragments in this disclosure against the in vitro proliferation inhibition of SK-BR-3 cells and U87 cells 50 value

[0828]

[0829] Conclusion: The small molecule fragments in this disclosure have obvious proliferation inhibition activities against SK-BR-3 cells and U87 cells, and the chiral center has a certain influence on the inhibitory activity of the compound.

[0830] Test Examples 1-2: In vitro proliferation inhibition test of the antibody-drug conjugate in this disclosure against tumor cells targeting HER2

[0831] The purpose of this experiment is to detect the inhibitory activity of the antibody-drug conjugate targeting HER2 in this disclosure against the in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, catalog number HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, catalog number HTB-132). Treat the cells with the compound at different concentrations in vitro, and after 6 days of culture, use CTG reagent to detect the cell proliferation. Evaluate the in vitro activity of the compound according to the IC 50 value.

[0832] According to 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, catalog number 16600-108) containing 10% FBS, EMEM medium (GE, catalog number SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, catalog number 11415-114) containing 10% FBS. The viable cell densities of the three cell lines were adjusted to 8.33×10 3 cells / mL, 8.33×10 3 cells / mL, and 1.39×10 4 cells / mL respectively using the cell culture media. After mixing the cell suspensions with adjusted densities, 180 μL of the cell suspension was added to each well of a 96-well cell culture plate. The relevant compounds were tested, and the results are shown in Table 2 below.

[0833] Table 2. IC 50 values of the in vitro proliferation inhibition of the tumor cells targeted by the antibody-drug conjugate of the present disclosure against HER2

[0834]

[0835] Conclusion: The antibody-drug conjugates of the present disclosure targeting the HER2 target have obvious proliferation inhibitory activity against HER2-positive cells SK-BR-3; at the same time, they have weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468 and have good selectivity.

[0836] Test Examples 1-3: Her2-ADC Plasma Stability Experiment

[0837] The ADC-19 sample, ADC-18 sample, ADC-20 sample, human plasma, monkey plasma (Shanghai Medicilon Inc.), and 1% BSA (Sigma) PBS solution (Shanghai Sangon Biotech Co., Ltd.) were filtered and sterilized using a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the above sterile plasma or 1% BSA PBS solution at a final concentration of 200 μg / mL, and incubated in a 37 °C cell culture incubator. The incubation day was recorded as Day 0, and then samples were taken on Days 7, 14, and 21 respectively for the detection of free toxins.

[0838] Take 25 μL of the sample into a 96-well plate; add 50 μL of the internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μL of acetonitrile; vortex mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and 5 μL was analyzed by LC / MS / MS (Applied Biosystems, USA).

[0839] Results showed that ADC-19 was quite stable in human and monkey plasma, as well as in 1% BSA PBS solution, with the release rate of free toxin not exceeding 2.1% at most, and it tended to be stable on the 14th day. See Figure 1A .

[0840] ADC-18 was poorly stable in human and monkey plasma, and the highest release rates of free toxin were 14.5% and 8.10% respectively. It was relatively stable in 1% BSA PBS solution. See Figure 1B .

[0841] ADC-20 was poorly stable in human plasma, monkey plasma and 1% BSA PBS solution, and the highest release rates of free toxin were 21.7%, 29.7% and 21.7% respectively. Moreover, it was in a degradation state in 1% BSA PBS solution all the time. See Figure 1C .

[0842] Test Example 1-4: Efficacy Evaluation of JIMT-1 Tumor-Bearing Mice

[0843] 1. Test Purpose

[0844] Using nunu nude mice as test animals, evaluate the efficacy of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 after intraperitoneal injection on nude mice transplanted with the human breast cancer cell trastuzumab (Trastuzumab) resistant strain (Herceptin) JIMT-1.

[0845] 2. Test Drugs and Materials

[0846] 2-1. Test Drugs

[0847] T-DM1 (prepared with reference to US20050169933)

[0848] ADC-21: 3 mg / kg

[0849] ADC-21: 10 mg / kg

[0850] ADC-24: 3 mg / kg

[0851] ADC-24: 10 mg / kg

[0852] Blank control (Blank): PBS

[0853] 2-2. Preparation Method: All were diluted and prepared with PBS.

[0854] 2-3. Test Animals

[0855] nunu nude mice, purchased from Vital River Laboratories (Beijing).

[0856] 3. Test Method

[0857] JIMT-1 cells (Nanjing Kebai) (5×10 6 / mouse, with 50% artificial basement membrane) were subcutaneously inoculated into the right costal region of mice, and the tumors grew for 8 days until they reached 203.09±11.94 mm 3 After that, the animals were randomly grouped (d1), with 8 animals per group and a total of 6 groups.

[0858] Intraperitoneal injection was used for drug administration, and the drug was administered 2 times in total. The tumor volume and body weight were measured 2 times a week, and the data were recorded.

[0859] Excel 2003 statistical software was used for data statistics: the average value was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV / SQRT; the P value of the difference between groups was calculated as TTEST.

[0860] The formula for calculating the tumor volume (V) is: V = 1 / 2×L 长 ×L 短 2

[0861] The relative tumor volume (RTV) = V T / V0

[0862] The tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[0863] where V0 and V T are the tumor volumes at the start and end of the experiment, respectively. C 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.

[0864] 4. Test results

[0865] The experimental results are as Figure 2 shown. After 2 intraperitoneal injections of the drug, the experiment was terminated on the 34th day of observation. T-DM1 (10 mg / kg) had no inhibitory effect on tumors; the tumor inhibition rate of ADC-21 at 3 mg / kg was 46.22% (P < 0.01); the tumor inhibition rate of ADC-21 at 10 mg / kg was 56.77% (P < 0.001); the tumor inhibition rate of ADC-24 at 3 mg / kg was 62.77% (P < 0.001); the tumor inhibition rate of ADC-24 at 10 mg / kg was 76.32% (P < 0.001). At the same dose, the tumor inhibition effect of ADC-24 was significantly better than that of ADC-21.

[0866] Test Examples 1 - 5: Pharmacodynamic Evaluation of SK-BR-3 Tumor-Bearing Mice

[0867] 1. Test purpose

[0868] Using nunu nude mice as test animals, evaluate the efficacy of Her2-ADC antibodies ADC-21 and ADC-22 after intraperitoneal injection on nude mice bearing xenografts of human breast cancer cell line SK-BR-3.

[0869] 2. Test drugs and materials

[0870] 2-1. Test drugs

[0871] ADC-21: 1 mg / kg

[0872] ADC-21: 6 mg / kg

[0873] ADC-22: 1 mg / kg

[0874] ADC-22: 6 mg / kg

[0875] Blank control: PBS.

[0876] 2-2. Preparation method: All are diluted and prepared with PBS.

[0877] 2-3. Test animals

[0878] nunu nude mice, purchased from Vital River Laboratories (Beijing).

[0879] 3. Test method

[0880] Inoculate SK-BR-3 cells (ATCC) (5×10 6 / mouse, with 50% artificial basement membrane) subcutaneously in the right rib region of mice. After the tumors grow for 20 days and reach 153.34 ± 11.73 mm 3 , randomly divide the animals into groups (d0), 8 animals / group, a total of 5 groups.

[0881] Administer the drug by intraperitoneal injection once. Measure the tumor volume and body weight twice a week and record the data.

[0882] Use Excel 2003 statistical software for data statistics: Calculate the average value as avg; Calculate the SD value as STDEV; Calculate the SEM value as STDEV / SQRT; Calculate the P value of the difference between groups as TTEST.

[0883] The formula for calculating the tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0884] Relative tumor volume (RTV) = V T / V0

[0885] Inhibitory rate (%) = (C RTV - T RTV ) / C RTV (%)

[0886] wherein V0 and V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the blank control and the experimental group at the end of the experiment, respectively.

[0887] 4. Test results

[0888] The experimental results are as Figure 3 shown. After a single intraperitoneal injection and observing until the 28th day to end the experiment, the inhibitory rate of ADC-21 at 1 mg / kg is 15.01%; the inhibitory rate of ADC-21 at 6 mg / kg is 77.4%, and there is a very significant difference compared with the blank control (P < 0.001). The inhibitory rate of ADC-22 at 1 mg / kg is 19.82%; the inhibitory rate of ADC-22 at 6 mg / kg is 98.38% (P < 0.001). At the same dose of 6 mg / kg, the inhibitory effect of ADC-22 is also significantly better than that of ADC-21.

[0889] Test Example 1-6: Plasma stability

[0890] The sample ADC-25 was mixed evenly with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100 μg / mL, filtered and sterilized, and then incubated in a 37°C water bath. The incubation day was recorded as day 0, and then samples were taken on days 7, 14, and 21 respectively for the detection of free toxin.

[0891] After the samples at different time points were taken out and placed at room temperature, they were vortexed and mixed evenly; 25 μL of the sample was taken into 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 and mixed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 5 μL of the supernatant was taken for LC / MS / MS analysis.

[0892] The results are as Figure 4 shown. ADC-25 is quite stable in human and monkey plasma, as well as 1% BSA PBS solution, and the release rate of free toxin does not exceed 2% at most, and tends to be stable on the 14th day.

[0893] Test Example 1-7: Efficacy evaluation of ADC on U87MG nude mouse xenografts of human brain astrocytoma

[0894] 1. Test purpose

[0895] In this experiment, BALB / cA-nude mice were used as test animals to evaluate the efficacy of the disclosed ADC compound against the xenograft tumors of human brain astrocytoma U87MG in nude mice.

[0896] 2. Test drugs and materials

[0897] 2-1. Test drugs

[0898] ADC-27 (3 mg / kg)

[0899] ADC-26 (3 mg / kg)

[0900] Blank control (Blank): PBS buffer at pH 7.4.

[0901] 2-2. Preparation method: PBS buffer at pH 7.4.

[0902] 2-3. Test animals

[0903] BALB / cA-nude mice: Purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0904] 3. Test methods

[0905] For the experimental BALB / cA-nude mice, female, 6-7 weeks old, human brain astrocytoma U87MG cells (human brain astrocytoma, Chinese Academy of Sciences Cell Bank, Catalog# TCHu138) were subcutaneously inoculated. On the tenth day after inoculating the cells, the animals were randomly grouped (D0), with 8 animals in each group, and started intraperitoneal injection once a week for a total of 3 times. The tumor volume and body weight were measured 2-3 times a week, and the data were recorded. The formula for calculating the tumor volume (V) is:

[0906] V = 1 / 2 × a × b 2

[0907] Where: a and b represent the length and width respectively.

[0908] Relative tumor volume (RTV) = V T / V0

[0909] Tumor inhibition rate (%) = (C RTV - T RTV ) / C RTV (%)

[0910] Where V0 and V T are the tumor volumes at the start and end of the experiment respectively. C RTV , T RTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment respectively.

[0911] 4. Test results

[0912] The drug was administered by intraperitoneal injection (i.p.) once a week for a total of 3 times. When observed until the 22nd day, the tumor inhibition rate of ADC-27 at 3 mg / kg reached 63.3% (P<0.0001); the inhibition rate of ADC-26 at 3 mg / kg reached 49.1%. ADC-27 showed stronger anti-tumor efficacy than ADC-26.

[0913] During the drug administration process, the body weights of the animals in each group were normal, indicating that ADC had no obvious toxic and side effects. The test results are shown in Table 3 and Figure 5 as follows. The antibodies tested could effectively inhibit the growth of U87MG xenografts in nude mice bearing tumors, and showed a dose-dependent manner.

[0914] Table 3. Efficacy of administered antibodies on U87MG xenografts in nude mice with human astrocytoma (D22)

[0915]

[0916] *** indicates P<0.001

[0917] Test Example 1-8: Evaluation of the Efficacy of ADC on Detroit 562 Xenografts in Nude Mice with Pleural Effusion Metastasis of Human Pharyngeal Cancer

[0918] 1. Test Purpose

[0919] In this experiment, BALB / cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compounds disclosed in this application on Detroit 562 xenografts in nude mice with pleural effusion metastasis of human pharyngeal cancer.

[0920] 2. Test Drugs and Materials

[0921] 2-1. Test Drugs

[0922] ADC-29 (3 mg / kg)

[0923] ADC-28 (3 mg / kg)

[0924] Negative control ADC (3 mg / kg): an antibody-drug conjugate formed by conjugating an antibody against a non-B7H3 target with Compound 20.

[0925] 2-2. Preparation Method: All were diluted and prepared with PBS.

[0926] 2-3. Test Animals

[0927] BALB / cA-nude nude mice: purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0928] 3. Test Method

[0929] BALB / cA-nude nude mice were used in the experiment. They were female, 6 - 7 weeks old, and were subcutaneously inoculated with human pharyngeal cancer pleural effusion metastatic cells Detroit 562 cells (ATCC, Catalog CCL-138 TM ). On the tenth day after inoculating the cells, the animals were randomly grouped (D0), with 8 animals in each group. Intraperitoneal injection of the drug was started once a week for a total of 3 times. The tumor volume and body weight were measured 2 - 3 times a week, and the data were recorded. The formula for calculating the tumor volume (V) is:

[0930] V = 1 / 2 × a × b 2

[0931] where: a and b represent the length and width respectively.

[0932] Relative tumor volume (RTV) = V T / V0

[0933] Tumor inhibition rate (%) = (C RTV - T RTV ) / C RTV (%)

[0934] where V0 and V T are the tumor volumes at the start and end of the experiment respectively. C 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.

[0935] 4. Test results

[0936] When intraperitoneal injection of the drug was given once a week for a total of 3 times and observed until the 28th day, the tumor inhibition rates of the tested ADCs were as follows: the tumor inhibition rate of ADC-29 at 3 mg / kg (3mpk) reached 72.27% (P < 0.001); the tumor inhibition rate of ADC-28 at 3 mg / kg (3mpk) reached 56.2% (P < 0.001). ADC-29 showed stronger anti-tumor efficacy than ADC-28 in all cases.

[0937] During the drug administration process, the body weights of the animals in each group were normal, indicating that ADC had no obvious toxic and side effects. The test results are shown in Table 4 and Figure 6 as follows. The detected antibodies could effectively inhibit the growth of Detroit 562 xenografts in tumor-bearing nude mice, and showed a dose-dependent manner.

[0938] Table 4. Efficacy of the administered antibody on Detroit 562 xenografts in tumor-bearing nude mice (D28)

[0939]

[0940] *** indicates P < 0.001

[0941] Test Examples 1 - 9: Efficacy Evaluation of U87 - MG Tumor - bearing Mice

[0942] 1. Test Objectives

[0943] Using Balb / c nude mice as test animals, evaluate the efficacy of B7H3 - antibody - drug conjugate after intraperitoneal injection in a human glioma cell U87MG xenograft tumor model.

[0944] 2. Test Drugs and Materials

[0945] 2 - 1. Test Drugs

[0946] ADC - 30 1mg / kg

[0947] ADC - 30 3mg / kg

[0948] ADC - 31 1mg / kg

[0949] ADC - 31 3mg / kg

[0950] Blank Control (Blank): PBS

[0951] 2 - 2. Preparation Method: All are diluted and prepared with PBS.

[0952] 2 - 3. Test Animals

[0953] BALB / cA - nude nude mice: Purchased from Shanghai Slac Laboratory Animal Co., Ltd.

[0954] 3. Test Methods

[0955] Inoculate U87MG cells (human brain astrocytoma, Chinese Academy of Sciences Cell Bank, Catalog#TCHu138) (2.5×10 6 / mouse) subcutaneously in the right rib of the mice. After the tumors grow for 14 days and reach 167.49mm 3 , randomly divide the animals into groups (d1), 8 animals / group, for a total of 5 groups.

[0956] Administer the drug by intraperitoneal injection once a week for a total of 3 times. Measure the tumor volume and body weight twice a week and record the data.

[0957] Use Excel 2003 statistical software for data statistics: The average value is calculated as avg; the SD value is calculated as STDEV; the SEM value is calculated as STDEV / SQRT; the P value of the difference between groups is calculated as TTEST.

[0958] The formula for calculating the tumor volume (V) is: V = 1 / 2×L 长 ×L 短2

[0959] Relative Tumor Volume (RTV) = V T / V0

[0960] Tumor Inhibition Rate (%) = (C RTV -T RTV ) / C RTV (%)

[0961] Where V0 and V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the vehicle control group and the experimental group at the end of the experiment, respectively.

[0962] 4. Test Results

[0963] The experimental results are as Figure 7 shown. When administered intraperitoneally once a week for a total of 3 times and observed until the 18th day, the tumor inhibition rates of the tested ADCs were as follows: the tumor inhibition rate of ADC-30 at 1 mg / kg was 0.31%; the tumor inhibition rate of ADC-30 at 3 mg / kg reached 45.23% (P < 0.0001); the tumor inhibition rate of ADC-31 at 1 mg / kg reached 39.22% (P < 0.01); the tumor inhibition rate of ADC-31 at 3 mg / kg reached 80.24% (P < 0.0001). At the same dose, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

[0964] II. Preparation Process Optimization

[0965] The exemplary products of the following examples have the structure shown by the following formula:

[0966]

[0967] Example 2-1

[0968] Under the condition of 0 °C, in 20 mM histidine-hydrochloric acid buffer (pH 5.6) containing 2.5 mM EDTA, 61.71 mg of trastuzumab antibody stock solution (0.0004251 mmol, diluted with 20 mM histidine-hydrochloric acid buffer to the final antibody concentration of 15 mg / mL) was stirred and reacted with 0.7311 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.002550 mmol) in a constant temperature water bath for 3 hours to generate intermediate I solution.

[0969] The shorter retention time compound 9-A (3.196 mg, 0.002975 mmol) of compound 9 was dissolved in 0.2062 mL of DMSO to form a DMSO solution of compound 9-A. 0.2052 mL of DMSO was pre-added to the above intermediate I solution, and then the DMSO solution of the above compound 9-A was added to the intermediate I solution pre-added with DMSO. The mixture was stirred at 25 °C in a water bath for 1 hour, and the reaction was quenched by adding an excess of cysteine. The exemplary product ADC-2-1 of the FADC-4A general formula was obtained.

[0970] The average value calculated by reverse phase chromatography-mass spectrometry: n = 5.45.

[0971] Example 2-2

[0972] Under the condition of 13 °C, in 20 mM histidine-hydrochloric acid buffer solution (pH 5.6) containing 2.5 mM EDTA, the stock solution of the antibody containing 61.71 mg of trastuzumab (0.0004251 mmol, the trastuzumab antibody was diluted with 20 mM histidine-hydrochloric acid buffer solution to the final antibody concentration of 15 mg / mL) and 0.5118 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001785 mmol) were stirred and reacted in a constant temperature water bath for 3 hours to form an intermediate I solution.

[0973] The shorter retention time compound 9-A (3.196 mg, 0.002975 mmol) of compound 9 was dissolved in 0.2062 mL of DMSO to form a DMSO solution of compound 9-A. 0.2052 mL of DMSO was pre-added to the above intermediate I solution, and then the DMSO solution of the above compound 9-A was added to the intermediate I solution pre-added with DMSO. The mixture was stirred at 25 °C in a water bath for 1 hour, and the reaction was quenched by adding an excess of cysteine. The exemplary product ADC-2-2 of the FADC-4A general formula was obtained.

[0974] The average value calculated by reverse phase chromatography-mass spectrometry: n = 5.49.

[0975] Example 2-3

[0976] Under the condition of 25 °C, in 20 mM histidine-hydrochloric acid buffer solution (pH 5.6) containing 2.5 mM EDTA, the stock solution of the antibody containing 61.71 mg of trastuzumab (0.0004251 mmol, the trastuzumab antibody was diluted with 20 mM histidine-hydrochloric acid buffer solution to the final antibody concentration of 15 mg / mL) and 0.4021 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001403 mmol) were stirred and reacted in a constant temperature water bath for 3 hours to form an intermediate I solution.

[0977] Dissolve the shorter retention time compound 9-A (3.196 mg, 0.002975 mmol) of Compound 9 in 0.2062 mL of DMSO to form a DMSO solution of Compound 9-A. Pre-add 0.2052 mL of DMSO to the above Intermediate I solution, then add the DMSO solution of the above Compound 9-A to the Intermediate I solution pre-added with DMSO, stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. The exemplary product ADC-2-3 of the FADC-4A general formula was obtained.

[0978] The average value calculated by reversed-phase chromatography-mass spectrometry: n = 5.39.

[0979] Example 2-4

[0980] Under the condition of 28 °C, in 20 mM histidine-hydrochloric acid buffer (pH 5.6) containing 2.5 mM EDTA, the stock antibody solution containing 61.71 mg of trastuzumab (0.0004251 mmol, diluted trastuzumab antibody with 20 mM histidine-hydrochloric acid buffer to the final antibody concentration of 15 mg / mL) and 0.3899 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001360 mmol) were stirred and reacted in a constant temperature water bath for 3 hours to form Intermediate I solution.

[0981] Dissolve the shorter retention time compound 9-A (3.196 mg, 0.002975 mmol) of Compound 9 in 0.2062 mL of DMSO to form a DMSO solution of Compound 9-A. Pre-add 0.2052 mL of DMSO to the above Intermediate I solution, then add the DMSO solution of the above Compound 9-A to the Intermediate I solution pre-added with DMSO, stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. The exemplary product ADC-2-4 of the FADC-4A general formula was obtained.

[0982] The average value calculated by reversed-phase chromatography-mass spectrometry: n = 5.44.

[0983] Example 2-5

[0984] Under the condition of 37 °C, in 20 mM histidine-hydrochloric acid buffer (pH 5.6) containing 2.5 mM EDTA, the stock antibody solution containing 61.71 mg of trastuzumab (0.0004251 mmol, diluted trastuzumab antibody with 20 mM histidine-hydrochloric acid buffer to the final antibody concentration of 15 mg / mL) and 0.3778 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001318 mmol) were stirred and reacted in a constant temperature water bath for 3 hours to form Intermediate I solution.

[0985] Dissolve the shorter retention time compound 9-A (3.196 mg, 0.002975 mmol) of compound 9 in 0.2062 mL of DMSO to generate a DMSO solution of compound 9-A. Add 0.2052 mL of DMSO to the above intermediate I solution in advance, and then add the DMSO solution of the above compound 9-A to the intermediate I solution with pre-added DMSO. Stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. Obtain the exemplary product ADC-2-5 of the general formula FADC-4A.

[0986] Calculated average value by reversed-phase chromatography-mass spectrometry: n = 5.46.

[0987] Example 2-6

[0988] Under the condition of 13 °C, in a 50 mM PBS buffer solution (pH 6.5) containing 2.5 mM EDTA, the antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol, dilute the trastuzumab antibody with 50 mM PBS buffer solution to the final antibody concentration of 15 mg / mL) and 0.4563 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001592 mmol) are stirred and reacted in a constant temperature water bath for 3 hours to generate an intermediate I solution.

[0989] Dissolve the shorter retention time compound 9-A (2.850 mg, 0.002653 mmol) of compound 9 in 0.1839 mL of DMSO to generate a DMSO solution of compound 9-A. Add 0.1829 mL of DMSO to the above intermediate I solution in advance, and then add the DMSO solution of the above compound 9-A to the intermediate I solution with pre-added DMSO. Stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. Obtain the exemplary product ADC-2-6 of the general formula FADC-4A.

[0990] Calculated average value by reversed-phase chromatography-mass spectrometry: n = 5.39.

[0991] Example 2-7

[0992] Under the condition of 25 °C, in a 50 mM PBS buffer solution (pH 6.5) containing 2.5 mM EDTA, the antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol, dilute the trastuzumab antibody with 50 mM PBS buffer solution to the final antibody concentration of 15 mg / mL) and 0.3477 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001213 mmol) are stirred and reacted in a constant temperature water bath for 3 hours to generate an intermediate I solution.

[0993] Dissolve the shorter retention time compound 9-A (2.850 mg, 0.002653 mmol) of Compound 9 in 0.1839 mL of DMSO to form a DMSO solution of Compound 9-A. Pre-add 0.1829 mL of DMSO to the above Intermediate I solution, and then add the DMSO solution of the above Compound 9-A to the Intermediate I solution pre-added with DMSO. Stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. The exemplary product ADC-2-7 of the FADC-4A general formula is obtained.

[0994] The average value calculated by reverse-phase chromatography-mass spectrometry: n = 5.50.

[0995] Example 2-8

[0996] Under the condition of 37 °C, in 50 mM PBS buffer (pH 6.5) containing 2.5 mM EDTA, the antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol, diluted with 50 mM PBS buffer to the final antibody concentration of 15 mg / mL) and 0.3259 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001137 mmol) are stirred and reacted in a constant temperature water bath for 3 hours to form Intermediate I solution.

[0997] Dissolve the shorter retention time compound 9-A (2.850 mg, 0.002653 mmol) of Compound 9 in 0.1839 mL of DMSO to form a DMSO solution of Compound 9-A. Pre-add 0.1829 mL of DMSO to the above Intermediate I solution, and then add the DMSO solution of the above Compound 9-A to the Intermediate I solution pre-added with DMSO. Stir and react at 25 °C in a water bath for 1 hour, and add an excess of cysteine to quench the reaction. The exemplary product ADC-2-8 of the FADC-4A general formula is obtained.

[0998] The average value calculated by reverse-phase chromatography-mass spectrometry: n = 5.47.

[0999] Example 2-9

[1000] Under the condition of 25 °C, in 20 mM histidine-hydrochloride buffer (pH 5.6) containing 2.5 mM EDTA, the antibody stock solution containing 127.4 g of trastuzumab (0.88 mmol, diluted with 20 mM histidine-hydrochloride buffer to the final antibody concentration of 15 mg / mL) and 0.83 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 2.90 mmol) are stirred and reacted in a constant temperature water bath for 3 hours to form Intermediate I solution.

[1001] Dissolve the shorter retention time compound 9-A (6.6 g, 6.14 mmol) of Compound 9 in 0.43 L of DMSO to form a DMSO solution of Compound 9-A. Add 0.43 L of DMSO to the above Intermediate I solution in advance, and then add the DMSO solution of the above Compound 9-A to the Intermediate I solution with pre-added DMSO. Stir and react at 25 °C in a water bath for 1 hour, and then stop the reaction.

[1002] Purify the above reaction solution with a Capto S Impact (GE) cation exchange chromatography column. Wash it with at least 9 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 elute with 0.05 M acetate buffer (pH 5.5, containing 0.39 M sodium chloride) to remove free toxins and residual solvents in the reaction solution. At 25 °C, perform 7-fold volume equal-volume ultrafiltration (30 kd ultrafiltration membrane package) on the cation eluate to change the solution to 0.01 M succinic acid buffer (pH 5.0) to obtain an exemplary product ADC-A1 of the general formula FADC-4A. Prepare 4 batches of samples by the above method.

[1003] The drug-loading amounts of the above 4 batches of samples were determined by reversed-phase chromatography-mass spectrometry to be 5.7. The yields of the four batches were 100.8%, 98.9%, 97.4% and 99.0% respectively.

[1004] Example 2-10

[1005] Under the condition of 37 °C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 3.55 mL, 35.5 μmol) to the PBS buffer solution of trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 164 mL, 11.08 μmol). Place it in a water bath oscillator and oscillate and react at 37 °C for 3.5 hours, and then stop the reaction. Cool the reaction solution to 25 °C with a water bath.

[1006] Dissolve the shorter retention time compound 9-A (185 mg, 172 μmol) of Compound 9 in 3.88 mL of acetonitrile and 1.94 mL of DMSO, add it to the above reaction solution, place it in a water bath oscillator, and oscillate and react at 25 °C for 3 hours, and then stop the reaction. Pass the reaction solution through an ultrafiltration membrane package and desalt and purify it successively with a PBS buffer aqueous solution (0.05 M PBS buffer aqueous solution with pH = 6.5) containing 2% (v / v) acetonitrile and 1% (v / v) DMSO and a succinic acid buffer aqueous solution (0.01 M succinic acid buffer aqueous solution with pH = 5.3) to remove small molecules to obtain a sample of an exemplary product ADC-B14 of the general formula FADC-4A, and store it at 4 °C.

[1007] Reverse-phase chromatography-mass spectrometry calculates the average value: n = 5.3.

[1008] Test Example 2-1 Drug Loading Distribution Test

[1009] 1. RP-DAR Determination Method

[1010] 1.1. RP-DAR analysis is carried out under the following measurement conditions:

[1011] UPLC system: Waters H-Class ultra-high performance liquid chromatography UPLC system

[1012] Detector: TUV detector (measurement wavelength: 280 nm)

[1013] Chromatographic column: Waters ACQUITY UPLC Protein BEH C4 (2.1 mm × 150 mm, 1.7 μm)

[1014] Column temperature: 80 °C

[1015] Flow rate: 0.3 mL / min

[1016] Sample chamber temperature: 20 °C

[1017] Run time: 25 min

[1018] Mobile phase A: 0.1% aqueous solution of difluoroacetic acid (DFA)

[1019] Mobile phase B: 0.1% DFA acetonitrile solution

[1020] Gradient program: 27.0% B - 44.0% B (0.00 min - 12.00 min), 44.0% B - 100% B (12.00 min - 13.00 min), 100% B - 100% B (13.00 min - 20.00 min), 100% B - 27.0% B (20.00 min - 20.04 min), 27.0% B - 27.0% B (20.04 min - 25 min)

[1021] Injected sample volume: 1.0 μL

[1022] 1.2. Data Analysis

[1023] When compared with the light chain (L0) and heavy chain (H0) of the antibody not bound to the drug, in the case of the light chain bound to the drug (one light chain bound to the drug: L1) and the heavy chain bound to the drug (one heavy chain bound to the drug: H1, two heavy chains bound to the drug: H2, three heavy chains bound to the drug: H3, four heavy chains bound to the drug: H4), the hydrophobicity increases in proportion to the number of drugs bound, and the retention time is prolonged. Therefore, elution is carried out in the order of L0, L1, H0, H1, H2, H3, and H4.

[1024] Since the drug linker absorbs UV, according to the following expression, using the molar extinction coefficients of the light chain, heavy chain, and drug linker, the obtained peak area is corrected according to the number of drugs bound. The calculation formula is as follows:

[1025] Light chain (εLC-280) / (εLC-280 + number of drugs linked × εdrug-280)

[1026] Heavy chain (εHC-280) / (εHC-280 + number of drugs linked × εdrug-280)

[1027] Note: εLC-280: Molar extinction coefficient of the light chain at 280 nm;

[1028] εHC-28: Molar extinction coefficient of the heavy chain at 280 nm;

[1029] εdrug-280: Molar extinction coefficient of the toxin at 280 nm.

[1030] Table 5. Calculation Table of Drug Loading Capacity in Reversed-Phase Chromatography

[1031] Name Number of conjugated drugs Corrected peak area percentage <![CDATA[L0]]> 0 <![CDATA[Peak area of 100×L0 correction / Total peak area of LC correction]]> <![CDATA[L1]]> 1 <![CDATA[100 × Peak area of L1 calibration / Total peak area of LC calibration]]> <![CDATA[H0]]> 0 <![CDATA[Peak area of 100×H0 calibration / Sum of peak areas of HC calibration]]> <![CDATA[H1]]> 1 <![CDATA[100 × Peak area of H1 calibration / Total peak area of HC calibration]]> <![CDATA[H2]]> 2 <![CDATA[100 × Peak area of H2 calibration / Total peak area of HC calibration]]> <![CDATA[H3]]> 3 <![CDATA[100 × Peak area of H3 correction / Total peak area of HC correction]]> <![CDATA[H4]]> 4 <![CDATA[Peak area of 100×H4 correction / Total peak area of HC correction]]>

[1032] Note: Total corrected peak area of LC = corrected peak area of L0 + corrected peak area of L1

[1033] Total corrected peak area of HC = corrected peak area of H0 + corrected peak area of H1 + corrected peak area of H2 + corrected peak area of H3 + corrected peak area of H4

[1034] Drug loading capacity n = 2 × Σ (number of drugs linked × percentage of corrected peak area) / 100

[1035] 2. Measurement Results

[1036] Table 6. Determination of Drug Loading Distribution

[1037]

[1038] Note: In ADC-2-1 to ADC-2-8, the content of H4 is below the detection limit (<<1%)

[1039] The results showed that for samples prepared with the same buffer system at different reduction reaction temperatures, the uniformity of the drug loading distribution of the samples increased significantly with the decrease of the reduction reaction temperature; for samples prepared with the same reduction reaction temperature but different buffer systems, the drug loading distribution of the samples prepared with the histidine-hydrochloric acid buffer system was more uniform.

[1040] Test Example 2-2: Free Toxin Test

[1041] 1. Method for Determining Free Toxin

[1042] 1.1. HPLC analysis was carried out under the following measurement conditions:

[1043] HPLC system: Waters H-Class ultra performance liquid chromatography UPLC system

[1044] Detector: TUV detector (measurement wavelength: 370 nm)

[1045] Chromatographic column: Waters ACQUITY UPLC Petide BEH C18( 2.1 mm × 150 mm, 1.7 μm)

[1046] Column temperature: 40 °C

[1047] Flow rate: 0.3 mL / min

[1048] Sample chamber temperature: 10 °C

[1049] Mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution

[1050] Mobile phase B: 0.1% TFA acetonitrile solution

[1051] Gradient program: 25.0% B - 25.0% B (0.00 min - 1.00 min), 25.0% B - 55.0% B (1.00 min - 17.00 min), 55.0% B - 25.0% B (17.00 min - 17.10 min), 25.0% B - 25.0% B (17.10 min - 20.00 min)

[1052] Injected sample volume: 5.0 μL

[1053] 1.2. Data analysis

[1054] The calculation formula for the LOD limit of the toxin is as follows:

[1055] Toxin limit (ppm) = 0.1 × 4 × 1000 / C

[1056] Note:

[1057] a) 0.1 is the concentration of the toxin LOD solution (μg / mL), 4 is the dilution factor during sample pretreatment, 1000 is the unit conversion factor, and C is the protein concentration of the sample being measured (mg / mlmL).

[1058] b) If the peak area of the free toxin in the test sample is less than the peak area of the LOD solution, it is determined to be less than the limit or not detected.

[1059] 2. Test results

[1060] Free toxin detection was performed on ADC-A1 (batches 1-4) and ADC-B14. The results (see Table 7) show that cation exchange column chromatography is not only suitable for large-scale preparation but also significantly reduces free toxin.

[1061] Table 7. Free toxin determination

[1062] Sample Drug loading Free toxin peak area ADC-A1 (Lot 1) 5.7 760 ADC-A1 (Lot 2) 5.7 123 ADC-A1 (Lot 3) 5.7 70 ADC-A1 (Lot 4) 5.7 37 ADC-B14 5.3 5423

Claims

1. A method for preparing an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is shown by the following formula: Wherein: n is from 3 to 8, and n is a decimal or an integer; The said preparation method comprises the following steps: Step (a): Reacting the antibody Trastuzumab with a reducing agent under the conditions of a reaction temperature of 1°C to 36°C and a pH of 5.0 to 6.0; Step (b): Reacting the product of step (a) with a compound shown by the following formula; 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. 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 claim 1, wherein the reaction temperature condition in step (a) is 25°C.

5. The method for preparing an antibody-drug conjugate according to claim 1, wherein the reaction in step (a) is carried out under the condition of a pH of 5.

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; the said buffer is a histidine salt buffer, a phosphate buffer or an acetate buffer.

7. The method for preparing an antibody-drug conjugate according to claim 6, wherein the said buffer is a histidine salt buffer containing EDTA.

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

9. The method for preparing an antibody-drug conjugate according to claim 1, wherein the said reducing agent in step (a) is tris(2-carboxyethyl)phosphine hydrochloride.

10. The method for preparing an antibody-drug conjugate according to claim 1, wherein the said preparation method further comprises step (c), and step (c) comprises purifying the product of step (b) by cation exchange chromatography or affinity chromatography.

11. The method for preparing an antibody-drug conjugate according to claim 10, wherein the said step (c) comprises subjecting the product of step (b) to cation exchange chromatography, and the packing material for the cation exchange chromatography is Capto S Impact or Poros XS.

12. The method for preparing an antibody-drug conjugate according to claim 10 or 11, wherein the packing material for the cation exchange chromatography is Capto S Impact.

13. The method for preparing an antibody-drug conjugate according to claim 1, wherein n is a decimal or an integer from 4 to 8.

14. The method for preparing an antibody-drug conjugate according to claim 1, wherein n is a decimal or an integer from 5 to 7.

15. The method for preparing an antibody-drug conjugate according to claim 1, wherein n is a decimal or an integer from 5.3 to 6.

1.

16. The method for preparing an antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate has a structure represented by the following formula: Among them, n is from 4 to 8, and n is a decimal or an integer; The said preparation method comprises the following steps: Step (a): Trastuzumab reacts with TCEP under the conditions that the reaction temperature is from 4°C to 30°C and the pH is from 5.0 to 6.0; Step (b): The product of step (a) reacts with a compound represented by the following formula; 17. The method for preparing an antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate has a structure represented by the following formula: Among them, n is from 4 to 8, and n is a decimal or an integer; The said preparation method comprises the following steps: Step (a): Trastuzumab reacts with TCEP under the conditions that the reaction temperature is 25°C and the pH is 5.6, and the said reaction is carried out in a histidine-hydrochloric acid buffer containing EDTA; Step (b): The product of step (a) reacts with a compound represented by the following formula; Step (c): It includes purifying the product of step (b) by cation exchange chromatography column.

18. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by the following formula: Among them, n is from 4 to 8, and n is a decimal or an integer; The said antibody-drug conjugate is prepared by the method for preparing an antibody-drug conjugate according to claim 17; and, the drug loading distribution of the said antibody-drug conjugate is: in the heavy chain population of the antibody, the proportion of the heavy chain of the antibody bound to 4 drugs is 4% or less, and the proportion of the heavy chain of the antibody not bound to the drug is 5% or less.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 18, wherein the drug loading distribution of the said antibody-drug conjugate is: in the heavy chain population of the antibody, the proportion of the heavy chain of the antibody bound to 4 drugs is 4% or less, and the proportion of the heavy chain of the antibody not bound to the drug is 5% or less; and in the light chain population of the antibody, the proportion of the light chain of the antibody bound to 1 drug is 65% or more.

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