HK40081719A - Preparation method for antibody medicament conjugate - Google Patents

Description

Preparation method of antibody drug conjugate

The present application claims priority from chinese patent application (application No. CN 202010219311.2) filed on 3/25/2020 and chinese patent application (application No. CN 202110297397.5) filed on 3/19/2021.

Technical Field

The present disclosure relates to a method for preparing a class of antibody drug conjugates, and more particularly, to a synthesis step and a purification step of a method for preparing a class of antibody drug conjugates.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Chemotherapy remains one of the most important anticancer modalities, including surgery, radiation therapy, and targeted therapies. Although the variety of highly effective cytotoxic drugs is large, the difference between tumor cells and normal cells is small, which limits the wide clinical application of these antitumor compounds due to toxic side effects. While the anti-tumor monoclonal antibody has specificity to the surface antigen of tumor cells, the antibody drug has become the front-line drug for anti-tumor therapy, but the curative effect is often not satisfactory when the antibody is used alone as the anti-tumor drug.

Antibody Drug Conjugate (ADC) connects monoclonal antibody or antibody fragment with cytotoxic drug with bioactivity via stable chemical linker compound, fully utilizes the specificity of antibody for binding normal cell and tumor cell surface antigen and high efficiency of cytotoxic drug, and avoids the defects of low curative effect of the former and excessive toxic and side effects of the latter. This means that the antibody Drug conjugate binds tumor cells precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs (Mullard A, (2013) Nature Reviews Drug Discovery,12: 329-332; DiJoseph JF, Armellono DC, (2004) Blood,103: 1807-1814).

The first antibody drug conjugate in 2000, Mylotarg (gemtuzumab ozogamicin, Hewlett packard, Inc.) was approved by the U.S. FDA for marketing for the treatment of acute myeloid leukemia (Drugs of the Future (2000)25(7): 686; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

In the year 2011, the month of 8,(Brentuximab vegatin, Seattle Gene genetics) for the treatment of Hodgkin lymphoma and recurrent anaplastic large cell lymphoma by the U.S. FDA Rapid evaluation channel (nat. Biotechnol (2003)21(7): 778-. Is a novel ADC drug, and can lead the drug to generate endocytosis after directly acting on a target spot CD30 on a lymphoma cell so as to induce the apoptosis of the tumor cell.

Both Mylotarg and Adcetris are targeted therapies against hematological tumors, which are relatively simple in tissue structure compared to solid tumors. In month 2 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) obtained U.S. FDA approval for the treatment of advanced or metastatic breast cancer patients who were positive for HER2 while being resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel (WO 2005037992; US 8088387). Kadcula is the first ADC drug approved by the FDA in the united states for the treatment of solid tumors.

There are several classes of cytotoxic small molecules for antibody drug conjugates, one of which is camptothecin derivatives, which have anti-tumor effects by inhibiting topoisomerase I. 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), is reported to be used in Antibody Drug Conjugates (ADCs) in WO 2014057687; clinical Cancer Research (2016)22 (20): 5097-5108; cancer Sci (2016)107: 1039-1046. There is still a need to develop more potent ADC drugs.

Disclosure of Invention

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) is as follows:

wherein:

w is selected from C _1-8 Alkyl radical, C _1-8 alkyl-C _3-7 Cycloalkyl or a linear heteroalkyl of 1 to 8 atoms containing 1 to 3 heteroatoms selected from N, O and S, wherein said C _1-8 Alkyl radical, C _3-7 Cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Substituted with one or more substituents in the cycloalkyl;

L ² is selected from-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-、-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-、-S(CH ₂ )p ¹ C (O) -and a chemical bond, wherein p ¹ Is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2 to 7 amino acid residues selected from the group consisting of amino acid residues consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Cycloalkyl substituted with one or more substituents;

R ¹ is halo C _1-6 Alkyl or C _3-7 A cycloalkyl group;

R ² selected from hydrogen atoms, halogeno-C _1-6 Alkyl and C _3-7 A cycloalkyl group;

or, R ¹ And R ² Together with the carbon atom to which they are attached form C _3-7 A cycloalkyl group;

R ⁵ selected from hydrogen atom, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

R ⁶ and R ⁷ Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

m is 0 or 1;

n is 3 to 8, n is a decimal or an integer;

pc is an antibody or antigen-binding fragment thereof;

the preparation method comprises the following steps:

step (a): reacting the antibody or antigen-binding fragment thereof with a reducing agent under reaction temperature conditions of about 1 ℃ to about 36 ℃;

step (b): reacting the product of step (a) with a compound represented by the following formula (La-Y-D);

wherein: w, L ² ，L ³ ，R ¹ ，R ² ，R ⁵ ，R ⁶ ，R ⁷ And m is as defined above.

In another aspect, the present disclosure provides a method of preparing an antibody drug conjugate having a structure represented by the following formula:

wherein n is 4 to 8, and n is a decimal or an integer;

the preparation method comprises the following steps:

step (a): reacting the antibody or antigen-binding fragment thereof with a reducing agent under reaction temperature conditions of about 1 ℃ to about 36 ℃;

Step (b): reacting the product of step (a) with a compound of the formula;

in alternative embodiments, the reaction temperature conditions in step (a) are from about 4 ℃ to about 30 ℃, preferably from about 20 ℃ to about 30 ℃, more preferably 25 ℃, non-limiting examples of which include about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃. In some embodiments, the reaction temperature conditions are 13 ℃ to 28 ℃ or 13 ℃ to 25 ℃.

In an alternative embodiment, the reaction in step (a) is carried out at a pH of from about 4.5 to about 6.5, preferably at a pH of from about 5.0 to about 6.0, more preferably at a pH of about 5.6. In non-limiting embodiments, 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.

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 the group consisting of histidine salt buffers, phosphate buffers and acetate buffers.

In an alternative embodiment, the reaction in step (a) is carried out in a buffer; in a non-limiting example, the buffer is histidine-hydrochloric acid buffer.

In alternative embodiments, the buffer is selected from the group consisting of EDTA-containing histidine salt buffers and EDTA-containing histidine-hydrochloric acid buffers. Illustratively, the concentration of the histidine salt buffer is from 1mM to 100mM, 10mM to 50mM, 20mM, 30mM or 40 mM; the concentration of EDTA is 1mM to 10mM, 2mM to 5mM, 2.5mM, 3mM or 4 mM. In some embodiments, the buffer comprises 10mM to 50mM histidine salt buffer and 1mM to 10mM EDTA. In some embodiments, the buffer comprises 20mM histidine-hydrochloric acid buffer and 2.5mM EDTA. EDTA refers to ethylenediaminetetraacetic acid.

In an alternative embodiment, said reducing agent in step (a) is selected from tris (2-carboxyethyl) phosphine (TCEP) or a salt thereof, 1, 4-Dimercaptothreitol (DTT) and β -mercaptoethanol (β -ME) and like suitable reducing agents, preferably TCEP or a salt thereof, more preferably tris (2-carboxyethyl) phosphine hydrochloride.

In alternative embodiments, the molar ratio of reducing agent to antibody or antigen-binding fragment thereof (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.

In an alternative embodiment, step (b) is carried out in an organic solvent, preferred organic solvents include dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), N-dimethylacetamide (DMAc), acetonitrile, or mixtures thereof. In a non-limiting embodiment, step (b) comprises: dissolving the compound represented by the formula (La-Y-D) in DMSO, and mixing the product of the step (a) with a DMSO solution of the compound represented by the formula (La-Y-D).

In an alternative embodiment, the above preparation method further comprises a step (c) comprising purifying the product of step (b). The purification can be carried out by cation column chromatography or affinity column chromatography, wherein the packing material for cation chromatography is selected from Capto S Impact and Poros XS, and Capto S Impact is preferred. In some embodiments, the Capto S Impact is Capto ^TM And S Impact. In some embodiments, the Poros XS is Poros ^TM XS。

In alternative embodiments, the drug load (n) may range from 3 to 8, 4 to 8, 5 to 7, preferably 5.3 to 6.1, more preferably 5.7 cytotoxic drugs per antibody or antigen binding fragment thereof (Pc). n is a decimal or an integer. In some embodiments, n is 5.3, 5.4, 5.5, 5.6, or 5.7.

In alternative embodiments, the antibody drug conjugate has a drug loading profile of: the proportion of antibody heavy chains that bind 4 drugs in the population of antibody heavy chains is 4% or less; preferably, the proportion of antibody heavy chains to which 4 drugs are bound is 4% or less, and the proportion of antibody heavy chains to which no drugs are bound is 6% or less. In non-limiting examples, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, 3% or less, 2% or less, or 1% or less; the proportion of the heavy chain of the antibody to which the drug is not bound is 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Alternatively, the proportion of antibody light chains that bind 1 drug in the population of antibody light chains 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 antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 65% or more. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 70% or more. In some embodiments, the antibody heavy chain proportion and/or the antibody light chain proportion is determined by reverse phase chromatography.

In an alternative embodiment, the preparation method is suitable for large scale preparation. The dosage of the antibody of the trastuzumab in the preparation method is more than 100mg, preferably more than 1 g, more preferably more than 10 g, and most preferably more than 100 g.

In an alternative embodiment, the antibody drug conjugate has the general formula (Pc-L) _b -Y-D) of the structure:

wherein:

s ¹ is an integer from 2 to 8;

Pc、R ¹ 、R ² 、R ⁵ 、R ⁶ 、R ⁷ m and n are as defined aboveDefining;

the preparation method comprises the following steps:

a step (a): reacting the antibody or antigen-binding fragment thereof with a reducing agent under reaction temperature conditions of about 1 to about 36 ℃;

a step (b): the product of step (a) is reacted with a compound of formula (L) _b -Y-D);

wherein: s is ¹ 、R ¹ 、R ² 、R ⁵ 、R ⁶ 、R ⁷ And m is as defined above.

In an alternative embodiment, the aforementioned antibody drug conjugate has the following structure:

wherein Pc and n are as defined in the general formula (Pc-La-Y-D).

In alternative embodiments, wherein the Pc is an antibody or antigen-binding fragment thereof, said antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, and a fully human antibody. In some embodiments, the antibody is a monoclonal antibody.

In alternative embodiments, the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-HER 2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-cl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-tescalin-C antibody, an anti-mescalin 44a4 antibody, and an anti-mesthelin antibody, or an antigen binding fragment thereof;

Preferably, the antibody or antigen-binding fragment thereof is selected from Trastuzumab (Trastuzumab), Pertuzumab (Pertuzumab), Nimotuzumab (Nimotuzumab), enobiluzumab, Emibetuzumab, Inotuzumab, pintuzumab, benztuximab (Brentuximab), Gemtuzumab (Gemtuzumab), Bivatuzumab, Lorvotuzumab, cBR96, and glemtuzumab, or an antigen-binding fragment thereof.

In alternative embodiments, the antibody conjugate has a structure represented by the formula:

wherein n is 4 to 8, and n is a decimal or an integer.

In another aspect, the present disclosure provides a method of preparing an antibody drug conjugate having a structure represented by the following formula:

wherein n is 4 to 8, and n is a decimal or an integer;

the preparation method comprises the following steps:

step (a): reacting Trastuzumab (Trastuzumab) with TCEP at a reaction temperature of about 4 ℃ to about 30 ℃ and a pH of about 4.5 to about 6.5;

a step (b): reacting the product of step (a) with a compound of the formula;

in alternative embodiments, the antibody drug conjugate has a structure represented by the formula:

wherein n is 4 to 8, and n is a decimal or an integer;

The preparation method comprises the following steps:

step (a): reacting Trastuzumab with TCEP at a reaction temperature of about 25 ℃ and a pH of about 5.6 in a histidine-hydrochloric acid buffer containing EDTA;

a step (b): reacting the product of step (a) with a compound of the formula;

step (c): comprising subjecting the product of step (b) to cation chromatography or affinity chromatography column purification. In some embodiments, the EDTA-containing histidine-hydrochloric acid buffer comprises 20mM histidine-hydrochloric acid buffer and 2.5mM EDTA.

The disclosure also provides an antibody drug conjugate or a pharmaceutically acceptable salt thereof, whereinThe structure of the antibody drug conjugate is shown as a general formula (Pc-L) _a -Y-D) represents:

wherein:

w is selected from C _1-8 Alkyl radical, C _1-8 alkyl-C _3-7 Cycloalkyl or a linear heteroalkyl of 1 to 8 atoms, said linear heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C is _1-8 Alkyl radical, C _3-7 Cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Cycloalkyl substituted with one or more substituents;

L ² Is selected from-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-、-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-、-S(CH ₂ )p ¹ C (O) -and a chemical bond, wherein p ¹ Is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residue is selected from the group consisting of amino acid residues of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), aspartic acid (D), and optionally further selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Substituted with one or more substituents in the cycloalkyl;

R ¹ is halo C _1-6 Alkyl or C _3-7 A cycloalkyl group;

R ² selected from hydrogen atoms, halogeno-C _1-6 Alkyl and C _3-7 A cycloalkyl group;

or, R ¹ And R ² Together with the carbon atom to which they are attached form C _3-7 A cycloalkyl group;

R ⁵ selected from hydrogen atoms, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

R ⁶ and R ⁷ Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

m is 0 or 1;

n is 4 to 8, n is a decimal or an integer;

pc is an antibody or antigen-binding fragment thereof;

the drug loading distribution of the antibody drug conjugate is as follows: the proportion of antibody heavy chains that bind 4 drugs in the population of antibody heavy chains is 4% or less; preferably, the proportion of antibody heavy chains to which 4 drugs are bound is 4% or less, and the proportion of antibody heavy chains to which no drugs are bound is 6% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind drugs is 4% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and the proportion of antibody light chains in the population of antibody light chains that bind 1 drug is 65% or more. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind drugs is 4% or less; and the proportion of antibody light chains in the population of antibody light chains that bind 1 drug is 70% or more.

The disclosure also provides an antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate is prepared by the preparation method of the antibody drug conjugate; and, the drug loading distribution of the antibody drug conjugate is: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less; preferably, the proportion of antibody heavy chains to which 4 drugs are bound is 4% or less, and the proportion of antibody heavy chains to which no drugs are bound is 6% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 65% or more. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 70% or more.

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:

wherein n is 4 to 8, and n is a decimal or an integer;

the antibody drug conjugate is prepared by the preparation method of the antibody drug conjugate; and, the drug loading distribution of the antibody drug conjugate is: the proportion of antibody heavy chains that bind 4 drugs in the population of antibody heavy chains is 4% or less; preferably, the proportion of antibody heavy chains to which 4 drugs are bound is 4% or less, and the proportion of antibody heavy chains to which no drugs are bound is 6% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 65% or more. In some embodiments, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains to which 4 drugs are bound is 1% or less, and the proportion of antibody heavy chains to which no drugs are bound is 4% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 70% or more.

Detailed Description

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

Term(s) for

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The present disclosure incorporates the entire contents of application PCT/CN2019/107873 into the present application.

An Antibody Drug Conjugate (ADC) is formed by connecting an antibody or an antibody fragment with a cytotoxin with biological activity or a small molecule drug with cell killing activity through a stable chemical linker compound, so that the specificity of the antibody to tumor cells or the specificity of high-expression antigen cell combination and the high efficiency of cytotoxin are fully utilized, and the toxic and side effects to normal cells are avoided. Compared with the traditional chemotherapy drugs, the antibody drug conjugate can accurately bind to tumor cells and reduce the influence on normal cells.

"buffering agent" refers to a buffering agent that is resistant to pH changes by the action of its acid-base conjugated components. Examples of buffers to control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.

A "histidine salt buffer" is a buffer comprising a histidine ion. Examples of the histidine salt buffer include buffers such as histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphoric acid, histidine-sulfuric acid, etc., preferably histidine-hydrochloric acid buffer, histidine-acetic acid buffer formulated with histidine and acetic acid, histidine-hydrochloric acid buffer formulated with histidine and hydrochloric acid or histidine and histidine hydrochloride.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid histidine salt, acetic acid-potassium acetate, acetic acid calcium acetate, acetic acid magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

The terms "about" and "approximately" as used herein mean a numerical value within an acceptable error range for the particular value determined by one of ordinary skill in the art, depending in part on how the value is measured or determined (i.e., the limits of the measurement system). For example, "about" in each practice in the art may mean within 1 or a standard deviation of more than 1. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean at most an order of magnitude or at most 5 times the value. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. diol. chem,243, p3558 (1968).

An "antibody" as described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure of a complete antibody composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The antibodies described in the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples being the following antibodies: one or more of an anti-HER 2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44a4 antibody, or an anti-sothelin antibody; preferred are trastuzumab (trade name: Herceptin), pertuzumab (trade name: pertuzumab, also known as 2C4, trade name: Perjeta), nimotuzumab (trade name: taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, phentuximab, gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and glemtuzumab.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in the disclosure conform in number and position to the known Kabat numbering convention (LCDR1-3, HCDR 1-3).

In the present disclosure, an antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

The term "murine antibody" in this disclosure is the preparation of an antibody from a mouse according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with a particular antigen and then isolating hybridomas that express antibodies having the desired sequence or functional properties.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. The construction of chimeric antibody includes the first establishing hybridoma secreting mouse-derived specific monoclonal antibody, the subsequent cloning of variable region gene from mouse hybridoma cell, the subsequent cloning of human antibody constant region gene, connecting the mouse variable region gene and human constant region gene into chimeric gene, inserting the chimeric gene into human carrier, and final expressing the chimeric antibody molecule in eukaryotic industrial system or prokaryotic industrial system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the strong heterologous reaction induced by the chimeric antibody because of carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database, as well as in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid decreased immunogenicity and, at the same time, decreased activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. Humanized antibodies of the disclosure also include humanized antibodies after further affinity maturation of the CDRs by phage display.

The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label.

An "antigen-binding fragment of an antibody" as described in the present disclosure may refer to a Fab fragment, a Fab 'fragment, a F (ab') ₂ Fragments, and Fv fragment scFv fragments that bind to an antigen. The Fv fragment contains the variable regions of the antibody heavy and light chains, but no constant regions, and has the smallest antibody fragment of the entire antigen-binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined into a single polypeptide chain using different linkers, known as single chain antibodies (scFv) or single chain fv (sFv).

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

"ADCC", which is antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity) as described in the present disclosure, refers to the direct killing of antibody-coated target cells by Fc-fragment recognition of an antibody by Fc-receptor expressing cells. ADCC effector function of an antibody may be reduced or eliminated by modification of the Fc portion of the IgG. The modification refers to mutation in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4 chimera, IgG 4F 234A/L235A mutation.

"mutations" in mutant sequences described in this disclosure include, but are not limited to, "back mutations," conservative modifications, "or" conservative substitutions or substitutions. "conservative modifications" or "conservative substitutions or substitutions" as referred to in the present disclosure refer to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.

The term "mutant sequence" as used herein refers to a nucleotide sequence and/or amino acid sequence having a different degree of percent sequence identity to the nucleotide sequence and/or amino acid sequence of the present disclosure, when mutational modifications such as substitutions, insertions or deletions are appropriately made to the nucleotide sequence and/or amino acid sequence of the present disclosure. The sequence identity recited in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and percent identity determination between two sequences can be performed by the default settings For the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.

The term "linker unit" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is linked at one end to an antibody or antigen-binding fragment thereof and at the other end to a drug, and may be linked to an antibody or drug after being linked to another linker.

Linkers, including extenders, spacers and amino acid units, may be synthesized by methods known in the art, such as those described in US2005-0238649A 1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research52: 127-.

Engineered antibodies or antigen-binding fragments of the disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting antibody fragments by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is immediately frozen, e.g., -70 ℃, or lyophilized.

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

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

The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH) ₂ -), 1-ethylidene (-CH (CH) ₃ ) -), 1, 2-ethylene (-CH) ₂ CH ₂ ) -, 1-propylene (-CH (CH) ₂ CH ₃ ) -), 1, 2-propylene (-CH) ₂ CH(CH ₃ ) -), 1, 3-propylene (-CH) ₂ CH ₂ CH ₂ -) 1, 4-butylene (-CH ₂ CH ₂ CH ₂ CH ₂ -) and 1, 5-butylene (-CH) ₂ CH ₂ CH ₂ CH ₂ CH ₂ -) and the like. The alkylene group may be substituted or unsubstituted and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally selected fromAlkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

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

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

The term "heterocyclyl" refers to a saturated or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O) _m (wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic radicals including spiro, fused and cyclic ringsAnd a heterocyclic group bridging the rings.

The term "spiroheterocyclyl" refers to 5-to 20-membered polycyclic heterocyclic groups in which one atom (referred to as a spiro atom) is shared between monocyclic rings, and in which one or more of the ring atoms is selected from nitrogen, oxygen, or S (O) _m (wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. The spiro heterocyclic group is classified into a single spiro heterocyclic group, a double spiro heterocyclic group or a multi spiro heterocyclic group according to the number of spiro atoms shared between rings, and preferably the single spiro heterocyclic group and the double spiro heterocyclic group. More preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl group. Non-limiting examples of spiro heterocyclyl groups include:

The term "fused heterocyclyl" refers to 5 to 20 membered polycyclic heterocyclic groups in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O) _m (wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

the term "bridged heterocyclyl" refers to 5-to 14-membered polycyclic heterocyclic groups in which any two rings share two atoms not directly attached, which may contain one atomOne or more double bonds, but not one ring having a completely conjugated pi-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S (O) _m (wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

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

and the like.

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

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

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

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

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

The term "amino protecting group" is intended to protect an amino group with a group that can be easily removed in order to keep the amino group unchanged when the rest of the molecule is subjected to a reaction. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl and the like. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.

The term "cycloalkylalkyl" means an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

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

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

The term "hydroxy" refers to an-OH group.

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

The term "amino" refers to-NH ₂ 。

The term "nitro" means-NO ₂ 。

The term "cyano" refers to — CN.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group having a free hydrogen may be unstable in combination with a carbon atom having an unsaturated (e.g., olefinic) bond.

The term "drug loading" or "drug loading" (DAR) refers to the average number of cytotoxic drugs loaded on each antibody or antigen-binding fragment thereof 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 embodiments of the present disclosure, the drug load is denoted as n, and may illustratively be the mean 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 spectroscopy, mass spectrometry, ELISA assays and HPLC.

The term "drug load distribution" refers to the distribution of antibodies linked to different numbers of drugs in a population of antibody drug conjugates, e.g., the distribution of antibodies linked to 0, 2, 4, 6, and 8 drugs in a population. Notably, DAR of 1, 3, 5 and 7 may also be included in the mixture due to the potential for degradation products. In the present disclosure, antibody drug load profiles can be characterized using antibody heavy chains that bind different amounts of drug, for example: h ₀ Represents the heavy chain of unbound drug, H ₁ Represents a heavy chain binding a drug, H ₂ Represents a heavy chain binding two drugs, H ₃ Represents the heavy chain binding three drugs, H ₄ Represents the heavy chain binding four drugs. Exemplary, H ₃ A ratio of 4% represents a ratio of 4% of the heavy chains binding three drugs in the heavy chain population of the antibody drug conjugate. Accordingly, antibody drug load profiles in the present disclosure can also be characterized using antibody light chains that bind varying amounts of drug, L ₀ Denotes the light chain of the antibody, L, which is not bound to the drug ₁ Representing the antibody light chain binding 1 drug.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, e.g., a composition comprising any one of the binding compounds of the present disclosure, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"substitution" refers to the replacement of a solvent system that solubilizes the antibody protein, e.g., by physically manipulating a high salt or hypertonic solvent system comprising the antibody protein using a buffer system of a stable formulation, such that the antibody protein is present in the stable formulation. Physical manipulations include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.

Drawings

FIG. 1A: results of plasma stability experiments for ADC-19 of the present disclosure.

FIG. 1B: the plasma stability test results for ADC-18 of the present disclosure.

FIG. 1C: results of plasma stability experiments for ADC-20 of the present disclosure.

FIG. 2: the present disclosure relates to evaluation of drug efficacy of ADC-21 and ADC-24 on JIMT-1 tumor-bearing mice.

FIG. 3: the ADC disclosed by the invention is used for evaluating the curative effect of human breast cancer cell SK-BR-3 transplantation tumor nude mice.

FIG. 4 is a schematic view of: results of plasma stability experiments for ADC-25 of the present disclosure.

FIG. 5 is a schematic view of: the efficacy of ADCs on human brain astrocytoma U87MG nude mouse transplantable tumors is disclosed.

FIG. 6: the ADC has the curative effect on human pharyngeal cancer hydrothorax metastatic cells Detroit 562 nude mouse transplantation tumor.

FIG. 7 is a schematic view of: the efficacy of ADCs to human glioma U87MG nude mouse transplantable tumors is disclosed.

Detailed Description

The present disclosure is further described in conjunction with the following examples, which are not intended to limit the scope of the present invention.

Experimental procedures, where specific conditions are not noted in the examples of the disclosure, are generally in accordance with conventional conditions, or in accordance with conditions recommended by the manufacturer of the raw materials or goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Preparation of antibody drug conjugate and property and biological test thereof

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl) ₃ ) Deuterated methanol (CD) ₃ OD), internal standard Tetramethylsilane (TMS), chemical shift is 10 ^-6 (ppm) is given as a unit.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

UPLC was measured using a Waters acquisition UPLC SQD LC Mass spectrometer.

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

UV-HPLC was measured using a Thermo nanodrop2000 ultraviolet spectrophotometer.

Proliferation inhibition rate and IC ₅₀ The values were determined with a PHERA starFS microplate reader (BMG, Germany).

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of Taiwan yellow sea as a carrier.

Known starting materials of the present disclosure may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & co.kg, Acros Organnics, Aldrich Chemical Company, shao zuo remote Chemical technology (Accela ChemBio Inc), dare chemicals, and the like.

In the examples, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

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

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction used a hydrogenation apparatus of Parr 3916EKX type and a hydrogen generator of Qinglan QL-500 type or a hydrogenation apparatus of HC2-SS type.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

In the examples, the reaction temperature was room temperature unless otherwise specified.

The room temperature is the optimum reaction temperature, and the temperature range is 20-30 ℃.

Preparation of PBS buffer at pH 6.5 in examples: take KH ₂ PO ₄ 8.5g，K ₂ HPO ₄ .3H ₂ Placing 8.56g of O, 5.85g of NaCl and 1.5g of EDTA in a bottle, fixing the volume to 2L, completely dissolving the components by ultrasonic waves, and shaking up to obtain the product.

The system of eluents for column chromatography and developing agents for thin layer chromatography used for purifying compounds include: a: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: the volume ratio of the solvent is adjusted according to different polarities of the compounds, and a small amount of triethylamine and acidic or basic reagents can be added for adjustment.

Some of the compounds of the disclosure were characterized by Q-TOF LC/MS. Q-TOF LC/MS used an Agilent 6530 accurate mass number quadrupole-time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra high performance liquid chromatograph (Agilent Poroshell 300 SB-C85 μm, 2.1X 75mm column).

Examples 1 to 1

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

To irinotecan mesylate 1b (2.0mg, 3.76 μmol, prepared as disclosed in the patent application "EP 0737686a 1") was added 1mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice-water bath, and a drop of triethylamine was added and stirred until the reaction solution became clear. 1-Hydroxycyclopropylcarboxylic acid 1a (1.4mg, 3.7. mu. mol, prepared by a known method "Tetrahedron Letters,25(12), 1269-72; 1984") and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (3.8mg, 13.7. mu. mol) were added to the reaction mixture in this order, and the reaction was stirred at 0-5 ℃ for 2 hours. To the reaction solution was added 5mL of water to quench the reaction, the reaction solution was extracted with ethyl acetate (8mL × 3), the organic phases were combined, washed with a saturated sodium chloride solution (5mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system B to give the title product 1(1.6mg, yield: 82.1%).

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

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

Examples 1 to 2

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

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

2mL of ethanol and 0.4mL of N, N-dimethylformamide were added to 1b (4mg, 7.53. mu. mol), replaced with argon three times, cooled to 0-5 ℃ in an ice-water bath, and 0.3mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3mg, 19.8. mu. mol, prepared by the method disclosed in patent application "WO 2013106717"), 1-hydroxybenzotriazole (3mg, 22.4. mu. mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.3mg, 22.4. mu. mol) were added to the reaction solution in this order at 0-5 ℃ after completion of additionThe reaction was stirred for 1 hour. The ice water bath was removed and the mixture was heated to 30 ℃ 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: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product (2-a: 1.5mg, 2-B: 1.5 mg).

MS m/z(ESI):534.0[M+1]。

Single configuration Compound 2-B (shorter Retention time)

UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.13(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H)。

Single configuration Compound 2-A (longer Retention time)

UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H)。

Examples 1 to 3

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

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

To 1b (5.0mg, 9.41. mu. mol) was added 2mL of ethanol and 0.4mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice-water bath, 0.3mL of N-methylmorpholine was added dropwise, and the reaction was stirred until the reaction became clear. To the reaction solution were added 3,3, 3-trifluoro-2-hydroxypropionic acid 3a (4.1mg, 28.4. mu. mol, supplier Alfa), 1-hydroxybenzotriazole (3.8mg, 28.1. mu. mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5.4mg, 28.2. mu. mol) in this order, and after completion of the addition, the reaction was stirred at 0 to 5 ℃ for 10 minutes. The ice water bath was removed and the mixture was heated to 30 ℃ and stirred for 8 hours. The reaction mixture 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 19X 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product (1.5mg ).

MS m/z(ESI):561.9[M+1]。

Single configuration compounds (shorter retention time)

UPLC analysis: retention time 1.11 min, purity: 88% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5.61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H), 2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。

Single configuration compounds (longer retention time)

UPLC analysis: retention time 1.19Minute, purity: 90% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,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)。

Examples 1 to 4

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

To 1b (3.0mg, 5.64. mu. mol) was added 1mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice water bath, and one drop of triethylamine was added thereto and stirred until the reaction solution became clear. To the reaction solution were added 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2mg, 16.9. mu. mol, prepared by the method disclosed in patent application "WO 2013106717") and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (4.7mg, 16.9. mu. mol) in this order, and after the addition, the reaction was stirred at 0 to 5 ℃ for 1 hour. The reaction solution was quenched with 5mL of water, extracted with ethyl acetate (10mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (5mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system B to give the title product 4(2.5mg, yield: 80.9%).

MS m/z(ESI):548.0[M+1]。

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

Examples 1 to 5

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

To 1b (2.0mg, 3.76. mu. mol) was added 1mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice-water bath, a drop of triethylamine was added dropwise, and the mixture was stirred until the reaction solution became clear. To the reaction solution were added 1- (hydroxymethyl) -cyclopentanecarboxylic acid 5a (0.87mg, 7.5. mu. mol, prepared by the method disclosed in patent application "WO 201396771") and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (2mg, 7.24. mu. mol) in this order, and after the addition, the reaction was stirred at 0-5 ℃ for 2 hours. To the reaction solution was added 5mL of water to quench the reaction, the reaction solution was extracted with ethyl acetate (8mL × 3), the organic phases were combined, washed with a saturated sodium chloride solution (5mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system B to give the title product 5(1.0mg, yield: 50%).

MS m/z(ESI):533.9[M+1]。

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

Examples 1 to 6

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

To 1b (3.0mg, 5.64. mu. mol) was added 1mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice-water bath, a drop of triethylamine was added dropwise, and the mixture was stirred until the reaction solution became clear. To the reaction solution were added 1- (hydroxymethyl) cyclobutane-1-carboxylic acid 6a (2.2mg, 16.9. mu. mol; prepared by the method disclosed in the 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-methylchloromorpholine salt (4.7mg, 16.9. mu. mol) in this order, and after completion of the addition, the reaction was stirred at 0 to 5 ℃ for 1 hour. To the reaction solution was added 5mL of water to quench the reaction, the reaction solution was extracted with ethyl acetate (10mL × 3), the organic phases were combined, washed with a saturated sodium chloride solution (5mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system B to give the title product 6(2.1mg, yield: 67.9%).

MS m/z(ESI):548.0[M+1]。

¹ H NMR(400MHz,DMSO-d ₆ ):δ7.85-7.62(m,1H),6.88(br，1H),5.87-5.48(m，2H),5.47-5.33(m，1H),5.31-5.06(m，1H),4.25-3.91(m,2H),3.25(br,1H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H)。

Examples 1 to 7

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

To 1b (3.0mg, 5.64. mu. mol) was added 2mL of ethanol and 0.4mL of N, N-dimethylformamide, cooled to 0-5 ℃ in an ice-water bath, 0.3mL of N-methylmorpholine was added dropwise, and the reaction mixture was stirred until the reaction solution became clear. To the reaction solution were added 1-hydroxycyclobutanecarboxylic acid 7a (2.0mg, 17.22. mu. mol, commercially available Yam), 1-hydroxybenzotriazole (2.3mg, 17.0. mu. mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.2mg, 16.7. mu. mol) in this order, and after completion of addition, the reaction was stirred at 0 to 5 ℃ for 10 minutes. The ice water bath was removed and 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 with developer system B to give the title product 7(2.5mg, yield: 83.1%).

MS m/z(ESI):534.0[M+1]。

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5.59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.15-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H),2.03-1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H)。

Examples 1 to 8

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

First step of

Benzyl 1- ((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methoxy) cyclopropane-1-carboxylate 8c

Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104mg, 0.54 mmol; prepared as disclosed in patent application "US 2005/20645") and 2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methyl acetate 8b (100mg, 0.27 mmol; prepared by the method disclosed in patent application "CN 105829346A") was added to a reaction flask, 5mL of tetrahydrofuran was added, argon was substituted three times, the temperature was lowered to 0-5 ℃ in an ice water bath, potassium tert-butoxide (61mg, 0.54mmol) was added, the ice bath was removed, the mixture was warmed to room temperature and stirred for 10 minutes, 20mL of ice water was added, extracted with ethyl acetate (5mL × 2) and chloroform (5mL × 5), the organic phases were combined and concentrated. The resulting residue was dissolved in 3mL of 1, 4-dioxane, 0.6mL of water was added, and sodium hydrogencarbonate (27mg, 0.32mmol) and 9-fluorenylmethyl chloroformate (70mg, 0.27mmol) were added and the mixture was stirred at room temperature for 1 hour. 20mL of water was added, extraction was performed with ethyl acetate (8 mL. times.3), the organic phase was washed with a saturated sodium chloride solution (20mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system B to give the title product 8c (100mg, yield: 73.6%).

MS m/z(ESI):501.0[M+1]。

Second step of

1- ((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methoxy) cyclopropane-1-carboxylic acid 8d

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

MS m/z(ESI):411.0[M+1]。

The third step

(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

Adding 1b (7mg, 0.013mmol) into a reaction bottle, adding 1mL of N, N-dimethylformamide, replacing with argon for three times, cooling to 0-5 ℃ in an ice-water bath, adding a drop of triethylamine, adding 8d (7mg, 0.017mmol) of 0.5mL of N, N-dimethylformamide solution, adding 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride (7mg, 0.026mmol), and stirring in an ice bath for reacting for 35 minutes. 10mL of water was added, extracted with ethyl acetate (5 mL. times.3), the organic phase was washed with saturated sodium chloride solution (10mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system B to give the title product 8e (8.5mg, 78.0% yield).

MS m/z(ESI):828.0[M+1]。

The fourth step

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

8e (4mg, 4.84. mu. mol) was dissolved in 0.2mL of dichloromethane, 0.1mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 2mL of toluene was added and concentrated under reduced pressure twice, 3mL of n-hexane was added and slurried, the upper n-hexane layer was decanted and repeated three times, and concentrated under reduced pressure to give the crude title product 8f (2.9mg) which was used in the next reaction without purification.

MS m/z(ESI):606.0[M+1]。

The fifth step

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

Crude 8f (2.9mg, 4.84. mu. mol) was dissolved in 0.5mL of N, N-dimethylformamide, replaced with argon three times, cooled in an ice-water bath to 0-5 ℃ and added with 8g (2.7mg, 5.80. mu. mol) of (S) -2(-2- (-2- (6- (2, 5-dioxo-1H-pyrrol-1-yl) hexanamido) acetamido) -3-phenylpropionic acid (0.3 mL of N, N-dimethylformamide solution prepared by the method disclosed in the patent application "EP 2907824"), added with 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (2.7mg, 9.67. mu. mol), stirred in an ice bath for 30 minutes, and the ice bath removed, warmed to room temperature and stirred for 15 minutes. Purifying the reaction solution by high performance liquid chromatography (separation conditions: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 8(2mg, yield: 39.0%).

MS m/z(ESI):1060.0[M+1]。

¹ H NMR(400MHz,DMSO-d ₆ ):δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H),4.52(t,1H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37-2.23(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H),1.33-1.11(m,5H),0.91-0.81(m,4H)。

Examples 1 to 9

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

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

First step of

2-cyclopropyl-2-hydroxyacetic acid benzyl ester 9a

2a (1.3g, 11.2 mmol; prepared by the method disclosed in patent application "WO 2013/106717") was dissolved in 50mL acetonitrile and potassium carbonate (6.18g, 44.8mmol), benzyl bromide (1.33mL, 11.2mmol) and tetrabutylammonium iodide (413mg, 1.1mmol) were added in that order. The reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (10mL), the combined filtrates were concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C to give the title product 9a (2g, yield: 86.9%).

Second step of

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

9a (120.9mg,0.586mmol) and 8b (180mg, 0.489mmol) were added to a reaction flask, 4mL of tetrahydrofuran was added, replaced with argon three times, the ice-water bath was cooled to 0-5 deg.C, potassium tert-butoxide (109mg, 0.98mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 40 minutes, 10mL of ice-water was added, extraction was performed with ethyl acetate (20 mL. times.2) and chloroform (10 mL. times.5), the organic phases were combined and concentrated. The resulting residue was dissolved in 4mL of dioxane, 2mL of water was added, sodium bicarbonate (49.2mg, 0.586mmol) and 9-fluorenylmethyl chloroformate (126mg, 0.49mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), and the organic phase was washed with a saturated sodium chloride solution (20mL), 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 developer system C to give the title product 9b (48mg, yield: 19%).

MS m/z(ESI):515.0[M+1]。

The third step

10-cyclopropyl-1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diaza undec-11-oic acid 9c

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

MS m/z(ESI):424.9[M+1]。

The fourth step

(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

Adding 1b (10mg, 18.8 mu mol) into a reaction bottle, adding 1mL of N, N-dimethylformamide, replacing with argon for three times, cooling to 0-5 ℃ in ice water bath, adding one drop of triethylamine, adding crude product 9c (13mg, 30.6 mu mol), adding 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methyl morpholine chloride salt (16.9mg, 61.2 mu mol), and stirring for reacting for 40 minutes in ice bath. 10mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 9d (19mg, yield: 73.6%).

MS m/z(ESI):842.1[M+1]。

The fifth step

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

9d (19mg, 22.6. mu. mol) was dissolved in 2mL of methylene chloride, 1mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, 1mL of toluene was added and concentrated under reduced pressure, and this was repeated twice. 3mL of n-hexane was added to the residue for beating, and after standing, the supernatant was poured off to retain the solid. The solid residue was concentrated under reduced pressure and oil-pumped to dryness to give the crude title product 9e (17mg), which was used in the next reaction without purification.

MS m/z(ESI):638.0[M+18]。

The sixth step

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.

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.

Crude 9e (13.9mg, 22.4. mu. mol) was dissolved in 0.6mL of N, N-dimethylformamide, replaced with argon three times, cooled in an ice-water bath to 0-5 ℃ and added with 8g (21.2mg, 44.8. mu. mol) of a 0.3mL solution of N, N-dimethylformamide, added with 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (18.5mg, 67.3. mu. mol), reacted with stirring in an ice bath for 10 minutes, removed from the ice bath, warmed to room temperature and stirred for 1 hour to give compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19X 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product (9-a: 2.4mg, 9-B: 1.7 mg).

MS m/z(ESI):1074.4[M+1]。

Single configuration compound 9-a (shorter retention time):

UPLC analysis, retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,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)。

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

UPLC analysis, retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H), 5.35-5.15(m,2H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H)。

Examples 1 to 10

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

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

First step of

3,3, 3-trifluoro-2-hydroxypropionic acid benzyl ester 10a

Dissolve 3a (1.80g, 12.5mmol) in 100mL acetonitrile and add potassium carbonate (5.17g, 37.5mmol), benzyl bromide (4.48mL, 37.5mmol) and tetrabutylammonium iodide (231mg, 0.63mmol) sequentially. The reaction solution was heated to 60 ℃ and stirred for 5 hours. The reaction solution was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C to give the title product 10a (980mg, yield: 33.5%).

¹ H NMR(400MHz,CDCl ₃ ):δ7.43-7.36(m,5H),5.34(s,2H),4.53(s,1H),3.44(s,1H)。

Second step of

Benzyl 1- (9H-fluoren-9-yl) -3, 6-dioxo-10- (trifluoromethyl) -2, 9-dioxa-4, 7-diaza-undec-11-oate 10b

Add 8b (63mg,0.17mmol) and 10a (80mg, 0.34mmol) to the flask, add 3mL tetrahydrofuran, replace three times with argon, cool the ice water bath to 0-5 deg.C, add potassium tert-butoxide (38mg, 0.34mmol), remove the ice bath, warm to room temperature and stir for 20 minutes, add 10mL ice water, extract with ethyl acetate (20 mL. times.2) and chloroform (10 mL. times.5), combine the organic phases and concentrate, dissolve the resulting residue in 2mL dioxane, add 0.4mL water, add sodium bicarbonate (19mg, 0.23mmol) and chloroformate-9-fluorenylmethyl ester (49mg, 0.19mmol), stir at room temperature for 1 hour. 20mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), the organic phase was washed with a saturated sodium chloride solution (20mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C to give the title product 10b (51mg, yield: 55.3%).

MS m/z(ESI):559.9[M+18]。

The third step

1- (9H-fluoren-9-yl) -3, 6-dioxo-10- (trifluoromethyl) -2, 9-dioxa-4, 7-diaza undec-11-oic acid 10c

10b (15mg, 0.28mmol) was dissolved in 3mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V: V ═ 2:1), palladium on carbon (15mg, content: 10%) was added, hydrogen gas was substituted three times, and the reaction was stirred at room temperature for 1 hour. The reaction was filtered through celite, the filter cake rinsed with tetrahydrofuran, and the filtrate concentrated to give the crude title product 10c (13 mg).

MS m/z(ESI):452.9[M+1]。

The fourth step

(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-oxoprop-2-yl) oxy) methyl) amino) -2-oxoethyl) carbamate 10d

Adding 1b (10mg, 18.8 mu mol) into a reaction bottle, adding 1mL of N, N-dimethylformamide, replacing with argon for three times, cooling to 0-5 ℃ in ice-water bath, adding one drop of triethylamine, adding 10c (13mg, 28.7 mu mol) of 0.5mL of N, N-dimethylformamide solution, adding 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methylmorpholine chloride salt (11mg, 39.7 mu mol), and stirring for reacting for 30 minutes in ice bath. 10mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), the organic phases were combined, the organic phase was washed with saturated sodium chloride solution (10 mL. times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system B to give the title product 10d (16mg, 97.8% yield).

MS m/z(ESI):870.0[M+1]。

The fifth step

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

10d (16mg, 18.4. mu. mol) was dissolved in 0.6mL of methylene chloride, and 0.3mL of diethylamine was added thereto and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 2mL of toluene was added and concentrated under reduced pressure, and this was repeated twice. Adding 3mL of n-hexane into the residue, pulping, standing, pouring out supernatant liquid, and keeping solid; this was repeated three times. The solid residue was concentrated under reduced pressure and oil pumped dry to give the crude title product 10e (12mg) which was used in the next reaction without purification.

MS m/z(ESI):647.9[M+1]。

The sixth step

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

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

Dissolve crude 10e (12mg, 18.5. mu. mol) in 1.0mL N, N-dimethylformamide, replace three times with argon, cool the bath to 0-5 ℃ with ice water, add 8g (14mg, 29.6. mu. mol) of 0.3mL N, N-dimethylformamide solution, add 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (15mg, 54.2. mu. mol), stir in ice bath for 30 minutes, remove the ice bath, warm to room temperature and stir for 1 hour to give compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19X 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc) B-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product (2.7mg, 2.6 mg).

MS m/z(ESI):1102.0[M+1]。

Single configuration compound (shorter retention time):

UPLC analysis: retention time 1.18 min, purity: 91% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,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)。

Single configuration compound (longer retention time):

UPLC analysis: retention time 1.23 min, purity: 90% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):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)。

Examples 1 to 11

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

First step of

Benzyl 1- ((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methoxy) cyclobutane-1-carboxylate 11b

1-Hydroxycyclobutanecarboxylic acid benzyl ester 11a (167mg, 0.81mmol, prepared by the method disclosed in the literature "Journal of Medicinal Chemistry,2013, vol.56, #13, p.5541-5552") and 8b (150mg, 0.41mmol) were added to a reaction flask, 5mL of tetrahydrofuran was added, argon was substituted three times, the ice-water bath was cooled to 0-5 ℃, potassium tert-butoxide (92mg, 0.82mmol) was added, the ice bath was removed, the mixture was stirred at room temperature for 10 minutes, 20mL of ice-water was added, extraction was performed with ethyl acetate (5 mL. times.2) and chloroform (5 mL. times.5), the organic phases were combined and concentrated, the resulting residue was dissolved in 3mL of dioxane, 0.6mL of water was added, sodium bicarbonate (41mg, 0.48mmol) and methyl chloroformate (105mg, 0.41mmol) and the mixture was stirred at room temperature for 1 hour. 20mL of water was added, extraction was performed with ethyl acetate (8 mL. times.3), the organic phase was washed with a saturated sodium chloride solution (20mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C to give the title product 11b (37mg, yield: 17.6%).

MS m/z(ESI):514.6[M+1]。

Second step of

1- ((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methoxy) cyclobutane-1-carboxylic acid 11c

11b (37mg, 71.9. mu. mol) was dissolved in 3mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V: V ═ 2:1), palladium on carbon (15mg, content: 10%) was added, hydrogen gas was substituted three times, and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered with celite, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the title product 11c (35mg, yield: 82%) which was directly used in the next step.

The third step

(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

Adding 1b (10mg, 0.018mmol) into a reaction flask, adding 1mL of N, N-dimethylformamide, replacing with argon three times, cooling to 0-5 ℃ in ice-water bath, adding one drop of triethylamine, adding 11c (13mg, 0.031mmol) of 0.5mL of N, N-dimethylformamide solution, adding 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride (25mg, 0.091mmol), and stirring for reacting for 40 minutes in ice bath. 8mL of water was added, extraction was performed with ethyl acetate (5 mL. times.3), the organic phase was washed with saturated sodium chloride solution (8mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system A to give the title product 11d (19mg, 73.9% yield).

MS m/z(ESI):842.3[M+1]。

The fourth step

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

11d (19mg, 22.6. mu. mol) was dissolved in 2mL of dichloromethane, 1mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 1mL of toluene was added and concentrated under reduced pressure twice, 4mL of n-hexane was added and slurried, the upper n-hexane layer was decanted and repeated three times, and concentrated under reduced pressure to give the crude title product 11e (15mg) which was used in the next reaction without purification.

The fifth step

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

Crude 11e (2mg, 3.22. mu. mol) was dissolved in 0.5mL of N, N-dimethylformamide, replaced with argon three times, cooled in an ice-water bath to 0-5 ℃ and 8g (1.5mg, 3.17. mu. mol) of a solution of 0.3mL of N, N-dimethylformamide was added, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (2.7mg, 9.67. mu. mol) was added and stirred at room temperature for 30 minutes. The reaction was pumped dry to remove DMF and the residue was taken up in DCM and purified directly by thin layer chromatography 2 times (polarity of developing solvent: DCM/MeOH: 10/1) to give the title product 11(1mg, yield: 28.8%).

MS m/z(ESI):1073.6[M+1]。

¹ H NMR(400MHz,CDCl ₃ ):δ8.70-8.60(m,1H),8.28-8.19(m,1H),8.13-7.91(m, 3H),7.79-7.71(d,1H),7.29(s,1H),7.25-7.09(m,4H),6.98(s,1H),6.71-6.62(m,1H),6.55-6.47(m,1H),5.64-5.54(m,2H),5.40(s,1H),5.35-5.27(t,2H),5.17-5.10(m,2H),4.60-4.51(m,1H),4.51-4.35(m,2H),3.93-3.78(m,3H),3.71-3.59(m,3H),3.01-2.88(m,3H),2.70-2.64(m,2H),2.44-2.30(m,3H),2.28-2.14(m,3H),2.11-1.92(m,6H),1.90-1.76(m,3H),1.51-1.39(m,4H),0.92-0.75(m,6H)。

Examples 1 to 12

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

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

First step of

3-cyclopropyl-2-hydroxypropionic acid 12b

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

Second step of

(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-hydroxypropionamide 12-A

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

To 1b (45mg, 0.085mmol) were added 1.5mL of ethanol and 1.5mL of N, N-dimethylformamide, replaced with argon three times, 0.1mL of N-methylmorpholine was added dropwise, and the reaction mixture was stirred until it became clear. To the reaction mixture were added 12b (90mg, 0.691mmol), 1-hydroxybenzotriazole (34mg, 0.251mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (49mg, 0.256mmol) in this order, and after completion of the addition, the reaction was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude compound 12 was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T C185 μm 21.2 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18mL/min) to give the title product (7mg, 15 mg).

MS m/z(ESI):547.9[M+1]。

Single configuration compounds (shorter retention time)

UPLC analysis: retention time 1.345 min, purity: 72% (column: ZORBAX eclipse Plus C181.8. mu.m 2.1X 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.42(d,1H),7.78(d,1H),7.30(s,1H),6.51(s,1H),5.60-5.50(m,2H),5.42(s,1H),5.19(q,2H),4.02-4.00(m,1H),3.21-3.11(m,2H),2.39(s,3H),2.21-2.07(m,2H),2.05-1.95(m,1H),1.92-1.68(m,4H),1.53-1.41(m,1H),0.87(t,3H),0.48-0.34(m,2H),0.14-0.01(m,2H)。

Single configuration compounds (longer retention time)

UPLC analysis: retention time 1.399 min, purity: 88% (column: ZORBAX eclipse Plus C181.8 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc),B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.36(d,1H),7.77(d,1H),7.31(s,1H),6.51(s,1H),5.58-5.51(m,1H),5.48(d,1H),5.42(s,1H),5.20(q,2H),4.09-4.02(m,1H),3.22-3.11(m,2H),2.39(s,3H),2.27-2.06(m,2H),2.05-1.95(m,1H),1.93-1.81(m,2H),1.65-1.43(m,2H),1.32-1.21(m,1H),0.87(t,3H),0.48-0.33(m,2H),0.14-0.01(m,2H)。

Examples 1 to 13 (reference example)

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

The title compound 13 is prepared by the method disclosed in "Example 76 on page 147 of the specification in EP2907824A 1".

Examples 1 to 14

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

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

First step of

3-cyclopropyl-2-hydroxypropionic acid benzyl ester 14a

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

Second step of

Benzyl 10- (cyclopropylmethyl) -1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-ate 14b

14a (94mg,0.427mmol) and 8b (130mg, 0.353mmol) were added to a reaction flask, 10mL of tetrahydrofuran was added, replaced with argon three times, the temperature in an ice-water bath was decreased to 0-5 ℃, potassium tert-butoxide (79mg, 0.704mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 10 minutes, 20mL of ice-water was added, extraction was performed with ethyl acetate (10 mL. times.4), the organic phase was washed with saturated sodium chloride solution (20mL), 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 the developer system C to give the title product 14b (50mg, yield: 26.8%).

MS m/z(ESI):529.2[M+1]。

The third step

10- (cyclopropylmethyl) -1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-oic acid 14c

14b (27mg, 0.051mmol) was dissolved in 3mL of ethyl acetate, palladium on carbon (7mg, content 10%, dry form) was added, hydrogen substitution was carried out three times, and the reaction was stirred at room temperature for 1 hour. The reaction was filtered through celite, the filter cake rinsed with ethyl acetate and the filtrate concentrated to give the crude title product 14c (23mg) which was directly used in the next reaction without purification.

MS m/z(ESI):439.1[M+1]。

The fourth step

(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

Add 1b (22mg, 42.38. mu. mol) to the flask, add 3mL of N, N-dimethylformamide, replace with argon three times, cool to 0-5 ℃ in an ice-water bath, add triethylamine (4.3mg, 42.49. mu. mol) dropwise, add crude 14c (23mg, 51.1. mu. mol), add 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (17.6mg, 63.6. mu. mol), stir in an ice bath for 40 minutes. 15mL of water was added, extraction was performed with ethyl acetate (8 mL. times.3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 14d (29mg, yield: 79.9%).

MS m/z(ESI):856.1[M+1]。

The fifth step

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

14d (29mg, 33.9. mu. mol) was dissolved in 0.8mL of methylene chloride, 0.4mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure, 1mL of toluene was added and concentrated under reduced pressure, and this was repeated twice. 3mL of n-hexane was added to the residue and slurried, and after standing, the supernatant was poured off and repeated three times. The residue was concentrated under reduced pressure and oil-pumped to dryness to give the crude title product 14e (22mg), which was used in the next reaction without purification.

MS m/z(ESI):634.1[M+1]。

The sixth step

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

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

Crude 14e (22mg, 33.9. mu. mol) was dissolved in 2.5mL of N, N-dimethylformamide, replaced with argon three times, cooled in an ice-water bath to 0-5 ℃ and added with 8g (24mg, 50.8. mu. mol) of 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (14mg, 50.6. mu. mol), the ice bath was removed, and the mixture was warmed to room temperature and stirred for 1 hour to give compound 14. The reaction mixture was purified by high performance liquid chromatography (separation conditions: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min) to give the title product (2mg ).

MS m/z(ESI):1088.4[M+1]。

Single configuration compound (shorter retention time):

UPLC analysis, retention time 1.18 min, purity: 88% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

Single configuration compound (longer retention time):

UPLC analysis, retention time 1.23 min, purity: 96% (column: ACQUITY UPLC BEHC 181.7 μm 2.1 x 50mm, mobile phase: A-water (5mmol NH) ₄ OAc), B-acetonitrile).

Examples 1 to 15

1- ((S) -9-benzyl-22- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -5,8,11,14, 17-pentaoxo-2-oxa-4, 7,10,13, 16-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.

First step of

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

Adding 8b (500mg, 1.35mmol) to a reaction flask, adding 6mL tetrahydrofuran, adding 1-hydroxymethylcyclopropane-1-carboxylic acid benzyl ester 15a (233mg, 1.13 mmol; prepared by the method disclosed in patent application "Example 22-2 on page 262 of the specification in EP2862856A 1) to the flask, replacing with argon three times, cooling in an ice-water bath to 0-5 ℃, adding sodium hydride (54mg, 1.35mmol), removing the ice bath, raising the temperature to room temperature and stirring for 40 minutes; to zero, 20mL of ice water was added, extraction was performed with ethyl acetate (5 mL. times.2) and chloroform (5 mL. times.5), the organic phases were combined, washed with saturated sodium chloride solution (20mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system B to give the title product 15B (15mg, yield: 2.5%).

MS m/z(ESI):515.2[M+1]。

Second step of

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

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

MS m/z(ESI):425.2[M+1]。

The third step

(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

Adding 1b (10mg, 0.021mmol) into a reaction bottle, adding 1mL of N, N-dimethylformamide, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, dropwise adding triethylamine, adding 15c (11mg, 0.026mmol), adding 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methylmorpholine chloride salt (10.7mg, 0.039mmol), and stirring at room temperature for reacting for 60 minutes after the addition. 10mL of water was added, extraction was performed with ethyl acetate (5 mL. times.3), the organic phase was washed with saturated sodium chloride solution (10mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system B to give the title product 15d (19mg, 87.0% yield).

MS m/z(ESI):842.2[M+1]。

The fourth step

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

15d (19mg, 22.56. mu. mol) was dissolved in 2mL of methylene chloride, 1mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. Concentrating the reaction solution at 0 ℃ under reduced pressure, adding 1mL of toluene, concentrating under reduced pressure, and repeating twice; adding 3mL of n-hexane for pulping, pouring out the n-hexane at the upper layer, and repeating for three times; concentration under reduced pressure gave the crude title product 15e (13.9mg), which was used in the next reaction without purification.

MS m/z(ESI):620.1[M+1]。

The fifth step

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.

Crude 15e (13.9mg, 22.4. mu. mol) was dissolved in 1mL of N, N-dimethylformamide under argonAfter three changes, the temperature in the ice-water bath was lowered to 0-5 ℃ and 8g (15.8mg, 33.4. mu. mol) of 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (9.3mg, 33.6. mu. mol) was added thereto, and the mixture was warmed to room temperature and stirred for 60 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19X 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 15(2.5mg, yield: 10.3%).

MS m/z(ESI):1074.2[M+1]。

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.51-8.37(m,1H),8.22(t,1H),8.14-8.02(m,2H),8.011-7.94(m,1H),7.82-7.73(m,1H),7.29(s,1H),7.26-7.10(m,3H),6.98(s,1H),6.53-6.47(m,1H),5.62-5.50(m,1H),5.45-5.36(m,1H),5.35-5.23(m,2H),5.13-5.02(m,2H),4.61-4.50(m,2H),4.42-4.28(m,2H),3.76-3.61(m,3H),3.60-3.45(m,3H),3.27-3.23(m,1H),3.20-2.81(m,7H),2.75-2.61(m,3H),241-2.28(m,3H),2.23-2.13(m,2H),2.11-2.01(m,1H),2.03-1.94(m,1H),1.90(s,1H),1.87-1.74(m,2H),1.53-1.36(m,3H),1.29-1.08(m,4H),0.90-0.68(m,4H)。

Examples 1 to 16

1- ((S) -9-benzyl-22- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -5,8,11,14, 17-pentaoxo-2-oxa-4, 7,10,13, 16-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) cyclobutane-1-carboxamide 16.

First step of

1- (hydroxymethyl) cyclobutane-1-carboxylic acid 16b

Ethyl 1- (hydroxymethyl) cyclobutanecarboxylate 16a (250mg, 1.58mmol, supplier Alfa) was dissolved in methanol (2mL) and water (1mL), sodium hydroxide (126mg, 3.15mmol) was added, the temperature was raised to 40 deg.C, and the reaction stirred for 3 hours. Cooled to room temperature, concentrated under reduced pressure to remove the organic solvent, back extracted with ether (10mL) and the aqueous phase collected. The aqueous phase was adjusted to pH 3-4 with 6N aqueous hydrochloric acid and concentrated under reduced pressure to give a solid. 3mL of toluene was added, concentrated under reduced pressure and spin-dried, and repeated three times. The oil pump was pulled dry to give the crude title product 16b (206mg), which was used in the next reaction without purification.

MS m/z(ESI，NEG):129.2[M-1]。

Second step of

Benzyl 1- (hydroxymethyl) cyclobutane-1-carboxylate 16c

Crude 16b (206mg, 1.58mmol) was dissolved in acetonitrile (15mL), anhydrous potassium carbonate (1.09g, 7.90mmol) and tetrabutylammonium iodide (29mg, 78.51. mu. mol) were added, benzyl bromide (216mg, 1.26mmol) was added, and the mixture was stirred at room temperature overnight. Filtration and concentration of the filtrate under reduced pressure, and purification of the resulting residue by silica gel column chromatography with developer system C gave the title product 16C (112mg, yield: 32.1%).

MS m/z(ESI):221.1[M+1]。

The third step

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

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

MS m/z(ESI):551.3[M+23]。

The fourth step

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

16d (12mg, 22.7. mu. mol) was dissolved in 1.5mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V: V ═ 2:1), palladium on carbon (5mg, content 10%) was added, hydrogen gas was substituted three times, and the reaction was stirred at room temperature for 2 hours. The reaction was filtered through celite, the filter cake rinsed with ethyl acetate, and the filtrate concentrated under reduced pressure to give the crude title product 16e (10mg) which was used in the next reaction without purification.

The fifth step

(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

Adding 1b (7.5mg, 0.014mmol) into a reaction bottle, adding 1mL of N, N-dimethylformamide, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, dropwise adding one drop of triethylamine, adding a solution of crude 16e (10mg) in 0.5mL of N, N-dimethylformamide, adding 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methylmorpholine chloride salt (6mg, 0.026mmol), and stirring in ice bath for reaction for 30 minutes. 10mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), the organic phase was washed with saturated sodium chloride solution (10mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system B to give the title product 16f (10.6mg, 87.8% yield).

MS m/z(ESI):856.2[M+1]。

The sixth step

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

16f (10.6mg, 12.4. mu. mol) was dissolved in 0.6mL of dichloromethane, 0.3mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. Concentrating the reaction solution under reduced pressure, adding 2mL of toluene, concentrating under reduced pressure, and repeating twice; 3mL of n-hexane was added for beating, and the upper n-hexane layer was decanted and repeated three times. Concentration under reduced pressure gave 16g (8mg) of the crude title product, which was used in the next reaction without purification.

MS m/z(ESI):634.1[M+1]。

Seventh step

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) cyclobutane-1-carboxamide 16.

16g (8mg) of the crude product was dissolved in 1mL of N, N-dimethylformamide, 8g (8.8mg, 18.6. mu. mol) was added, and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (5.2mg, 18.8. mu. mol) was added, and the reaction was stirred at room temperature for 30 minutes. The reaction mixture was purified by high performance liquid chromatography (separation conditions: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min) to give the title product 16(1.0mg, yield: 7.2%).

MS m/z(ESI):1088.0[M+1]。

Examples 1 to 17

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

First step of

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

1-phenyl-2, 5,8,11,14,17,20,23, 26-nonaoxadioctadecyl-28-ol 17a (0.34g, 0.67mmol, obtained from the supplier) was dissolved in 10mL of dichloromethane, and silver oxide (0.24g, 1.01mmol), t-butyl bromoacetate (0.16g, 0.81mmol) and potassium iodide (0.07g, 0.40mmol) were added in this order, followed by stirring at room temperature for reaction for 3 hours. Filtration and concentration of the filtrate under reduced pressure, and purification of the resulting residue by silica gel column chromatography using developer system B gave the title product 17B (0.42g, yield: 100%).

MS m/z(ESI):636.3[M+18]。

Second step of

29-hydroxy-3, 6,9,12,15,18,21,24, 27-nonaxomontan-1-carboxylic acid tert-butyl ester 17c

17b (417mg, 0.67mmol) was dissolved in 15mL of tetrahydrofuran, palladium on carbon (110mg, 10% content, dry form) was added, replaced with hydrogen three times, and the reaction was stirred at 60 ℃ for 3 hours. The reaction solution was filtered through celite, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give crude title product 17c (357mg), which was used in the next reaction without purification.

MS m/z(ESI):546.2[M+18]。

The third step

29-azido-3, 6,9,12,15,18,21,24, 27-nonaxomontanyl-1-tert-butyl ester 17d

17c (357mg,0.675mmol) was dissolved in 10mL of toluene, diphenyl azidophosphate (279mg, 1.014mmol) and 1, 8-diazabicycloundec-7-ene (206mg, 1.353mmol) were added, replaced with argon three times, the reaction was stirred at room temperature for 2 hours, and then warmed to 105 ℃ for 19 hours. The reaction mixture was cooled to room temperature, concentrated, 20mL of water was added, extracted with ethyl acetate (10 mL. times.4), and the organic phase was washed with a saturated sodium chloride solution (20mL), 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 developer system B to give crude title product 17d (412 mg).

MS m/z(ESI):571.3[M+18]。

The fourth step

29-amino-3, 6,9,12,15,18,21,24, 27-nonaxomontanyl-1-tert-butyl ester 17e

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

MS m/z(ESI):528.2[M+1]。

The fifth step

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-azatriundec-31-carboxylic acid tert-butyl ester 17f

(1r,4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxylic acid (98.5mg, 0.415mmol) was dissolved in 10mL of dichloromethane, 2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (190mg, 0.500mmol) and N, N-diisopropylethylamine (162mg, 1.253mmol) were added, argon was substituted three times, crude 17e (220mg, 0.417mmol) was added, and the reaction was stirred at room temperature for 1 hour. 15mL of water was added, extracted with dichloromethane (8 mL. times.3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15mL), the organic phase was 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 developer system B to give the title product 17f (122mg, yield: 39.2%).

MS m/z(ESI):747.2[M+1]。

The sixth step

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-azatriundec-31-oic acid 17g

17f (122mg, 0.163mmol) was dissolved in 0.8mL of dichloromethane, 0.4mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature for 1 hour. Adding 15mL of dichloromethane for dilution, and concentrating under reduced pressure; adding 10mL of normal hexane, concentrating under reduced pressure, and repeating twice; then 10mL of toluene is added and the mixture is concentrated under reduced pressure; with 10mL of n-hexane: the mixed solvent of ether 5:1 was slurried three times to pH near 7, concentrated, and pumped to dryness to give the title product 17g (98mg, yield: 86.8%).

MS m/z(ESI):691.2[M+1]。

Seventh step

2, 4-Dimethoxybenzyl 1- ((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methoxy) cyclopropyl-1-carboxylate 17H

8d (164mg, 0.40mmol) was dissolved in methylene chloride (5mL), and 2, 4-dimethoxybenzyl alcohol (81mg, 0.48mmol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (115mg, 0.60mmol) and 4-dimethylaminopyridine (5mg, 0.041mmol) were sequentially added to the solution, and after the addition was completed, the reaction was stirred at room temperature for 1 hour. 20mL of water was added, the layers were separated after shaking, the aqueous phase was extracted with dichloromethane (8 mL. times.3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (20mL), the organic phase was 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 developer system C to give the title product 17h (124mg, yield: 55.4%).

MS m/z(ESI):583.1[M+23]。

The eighth step

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-pentaazepin-17-yl) oxy) cyclopropyl-1-carboxylate 17j

17h (39mg, 69.6. mu. mol) was dissolved in 0.6mL of dichloromethane, 0.3mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. Concentrating the reaction solution under reduced pressure, adding 2mL of toluene, concentrating under reduced pressure, and repeating twice; adding 3mL of n-hexane for pulping, pouring out the n-hexane at the upper layer, repeating the steps for three times, and concentrating under reduced pressure. The resulting crude product was dissolved in 2mL of N, N-dimethylformamide, charged with (((9H-fluoren-9-yl) methoxy) carbonyl) glycyl-L-phenylalanine 17i (35mg, 69.8. mu. mol, prepared by the method disclosed in patent application "CN 108853514A, page 13, examples 7-12"), and added with 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (23mg, 83.1. mu. mol), and stirred at room temperature for 1 hour. 10mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10 mL. times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 17j (48mg, yield: 83.9%).

MS m/z(ESI):822.0[M+1]。

The ninth step

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

17j (48mg, 58.4. mu. mol) was dissolved in 1.4mL of a 3% (v/v) dichloromethane solution of dichloroacetic acid, cooled to 0-5 ℃ in an ice water bath, triethylsilane (21mg, 180.6. mu. mol) was added, and the reaction was stirred in an ice bath for 3 hours. Half of the organic solvent was removed by concentration under reduced pressure in an ice bath, 5mL of diethyl ether was added, the mixture was naturally warmed to room temperature and slurried, a white solid was precipitated, filtered, and the cake was collected and pumped to dryness by an oil pump to give the title product 17k (33mg, yield: 84.1%).

The tenth step

(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-tetraazatriden-13-yl) carbamate 17l

Adding 1b (20mg, 42.4 mu mol) into a reaction bottle, adding 1mL of 10% (v/v) methanol dichloromethane solution, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, dropwise adding a drop of triethylamine, and stirring until 1b is dissolved. 17k (33mg, 49.1. mu. mol) was dissolved in 1mL of a 10% (v/v) solution of methanol in methylene chloride, and then added dropwise to the above reaction solution, followed by addition of 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (17.6mg, 63.6. mu. mol). The reaction was warmed to room temperature and stirred for 1 hour. Adding 10mL of dichloromethane and 5mL of water, stirring for 5 minutes, standing for layering, and collecting an organic phase; the aqueous phase was extracted with dichloromethane (10 mL. times.3) and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10 mL. times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 17l (37mg, yield: 80.2%).

MS m/z(ESI):1090.1[M+1]。

The eleventh step

(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-nonaxo-2, 5,8,11, 14-pentaazatetratridec-43-yl) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxamide 17

17l (15.5mg, 14.23. mu. mol) was dissolved in 0.6mL of methylene chloride, and 0.3mL of diethylamine was added thereto and stirred at room temperature for 1.5 hours. Concentrating the reaction solution under reduced pressure, adding 2mL of toluene, concentrating under reduced pressure, and repeating twice; 3mL of n-hexane was added for beating, the upper n-hexane layer was decanted and repeated three times. Concentrated under reduced pressure and then pumped dry with an oil pump. The resulting crude product was dissolved in 1mL of N, N-dimethylformamide, 17g (11mg, 15.92. mu. mol) was added, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (6.0mg, 21.68. mu. mol) was added, and the mixture was replaced with argon three times, followed by stirring at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19X 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 17(6mg, yield: 27.4%).

MS m/z(ESI):1556.4[M+18]。

¹ H NMR(400MHz,DMSO-d ₆ ):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)。

Examples 1 to 18

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

First step of

(R) -2-cyclopropyl-2-hydroxyacetic acid benzyl ester 18a

(S) -2-cyclopropyl-2-hydroxyacetic acid benzyl ester 18b

Dissolve 2a (7.4g, 63.7mmol) in 200mL acetonitrile and add potassium carbonate (35g, 253.6mmol), benzyl bromide (9.3g, 54.4mmol) and tetrabutylammonium iodide (500mg, 1.36mmol) sequentially. The reaction solution was stirred at room temperature for 16 h, filtered through celite, the filter cake rinsed with ethyl acetate (10mL), the combined filtrates concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C, 4.1g, and further purified by chiral resolution, to give the title products 18a (1.1g) and 18b (1.2 g).

Second step of

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

Dissolve 8b (3.1g, 8.41mmol) in tetrahydrofuran (55mL), add 18a (2.0g, 9.70mmol), cool to 0-5 ℃ in an ice water bath, add potassium tert-butoxide (1.89g, 16.84mmol), and stir for 10 minutes in an ice water bath. Ethyl acetate (30mL) and water (20mL) were added, the layers were separated by standing, the aqueous layer was extracted with chloroform (30 mL. times.5), and the organic layers were combined. The organic phase was concentrated under reduced pressure, and the resulting residue was dissolved in 1, 4-dioxane (32mL) and water (8mL), and sodium carbonate (1.78g, 16.79mmol) and 9-fluorenylmethyl chloroformate (2.18g, 8.42mmol) were added and stirred at room temperature for 2 hours. Water (30mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL. times.3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography with developer system C to give the title product 18C (1.3g, yield: 30.0%).

MS m/z(ESI):515.2[M+1]。

The third step

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

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

MS m/z(ESI):425.1[M+1]。

The fourth step

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

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

MS m/z(ESI):575.0[M+1]。

The fifth step

2, 4-Dimethoxybenzyl (R) -2- ((2-Aminoacetylamino) methoxy) -2-cyclopropylacetate 18f

18e (33mg, 57.4. mu. mol) was dissolved in 0.6mL of methylene chloride, 0.3mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. Concentrating the reaction solution under reduced pressure, adding 2mL of toluene, concentrating under reduced pressure, and repeating twice; 3mL of n-hexane was added for slurrying, the upper n-hexane layer was decanted, repeated three times, and concentrated under reduced pressure to give the crude title product 18f (21mg) which was used in the next reaction without purification.

The sixth step

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-pentaazaeicosa-20-oate 18g

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

MS m/z(ESI):853.0[M+18]。

Seventh step

(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-pentaazaeicosa-20-oic acid 18H

18g (37mg, 44.3. mu. mol) of the mixture was dissolved in 1.4mL of a 3% (v/v) dichloromethane solution of dichloroacetic acid, cooled to 0-5 ℃ in an ice-water bath, triethylsilane (15.4mg, 132.4. mu. mol) was added, and the reaction was stirred in an ice bath for 3 hours. Half of the organic solvent was removed by concentration under reduced pressure in an ice bath, 5mL of diethyl ether was added, the mixture was naturally warmed to room temperature and slurried, a white solid was precipitated, filtered, and the cake was collected and pumped to dryness to obtain the title product 18h (24mg, yield: 79.1%).

MS m/z(ESI):708.2[M+23]。

Eighth step

(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

Adding 1b (30mg, 63.6 mu mol) into a reaction bottle, adding 1mL of 10% (v/v) methanol dichloromethane solution, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, adding a drop of triethylamine, and stirring until 1b is dissolved. 18h (65mg, 94.8. mu. mol) was dissolved in 1mL of a 10% (v/v) solution of methanol in dichloromethane, and then added dropwise to the above reaction solution, followed by addition of 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (27mg, 97.6. mu. mol). The reaction was warmed to room temperature and stirred for 1 hour. Adding 10mL of dichloromethane and 5mL of water, stirring for 5 minutes, standing for layering, and collecting an organic phase; the aqueous phase was extracted with dichloromethane (10 mL. times.3) and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 18i (25mg, yield: 35.6%).

MS m/z(ESI):1104.4[M+1]。

The ninth step

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

18i (12mg, 10.9. mu. mol) was dissolved in 0.6mL of dichloromethane, 0.3mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 2mL of toluene was added and concentrated under reduced pressure twice, 3mL of n-hexane was added and slurried, the upper n-hexane layer was decanted and repeated three times, and concentrated under reduced pressure to give the title product, 18j (10mg), as a crude product, which was used in the next reaction without purification.

MS m/z(ESI):881.0[M+1]。

The tenth step

(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-hexaoxy-3, 20,23,26,29,32,35,38,41, 44-decaoxa-5, 8,11,14, 17-pentaazatetrahexadec-46-yl) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxamide 18

Crude 18j (10mg) was dissolved in 1mL of N, N-dimethylformamide, 17g (8.5mg, 12.3. mu. mol) was added, and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (4.6mg, 16.6. mu. mol) was added and stirred at room temperature for 30 minutes. The reaction mixture was filtered and purified by HPLC (separation conditions: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 18(9.5mg, yield: 56.2%).

MS m/z(ESI):1570.2[M+18]。

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.77(d,1H),8.59-8.55(m,1H),8.42(d,1H),8.37-8.28(m,1H),8.25-8.06(m,2H),7.96-7.86(m,1H),7.86-7.70(m,2H),7.32-7.28(m,1H),7.25-7.14(m,3H),6.67(m,1H),5.96(s,1H),5.80-5.72(m,1H),5.62-5.52(m,2H),5.43-5.30(m,3H),5.28-5.17(m,2H),5.12-5.08(m,1H),4.72-4.35(m,8H),3.95-3.70(m,13H),3.35-3.22(m,14H),2.42-2.32(m,3H),2.05-1.98(m,4H),1.88-1.82(m,12H),1.47-1.39(m,3H),1.32-1.18(m,11H),0.90-0.80(m,4H),0.52-0.37(m,3H),0.32-0.18(m,2H)。

Examples 1 to 19

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

First step of

(S) -10-cyclopropyl-1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diaza-undec-11-oic acid benzyl ester 19a

Adding 18b (252mg,1.22mmol) into a reaction bottle, adding 4mL dichloromethane, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, adding lithium tert-butoxide (98mg,1.22mmol), stirring in the ice water bath for reaction for 15 minutes, clarifying, adding 8b (300mg, 814.3. mu. mol), and stirring in the ice water bath for 2.5 hours. Water (10mL) was added, the layers were separated, the aqueous layer was extracted with dichloromethane (8 mL. times.2), the organic layers were combined and washed with water (10 mL. times.1), saturated brine (10 mL. times.2), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The resulting residue was purified by silica gel column chromatography with developer system C to give the title product 19a (282mg, yield: 67.2%).

Second step of

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

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

The third step

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

Crude 19b (230mg, 541.8. mu. mol) was dissolved in 7mL of dichloromethane, and 2, 4-dimethoxybenzyl alcohol (136.7mg, 812.7. mu. mol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (155mg, 808.5. mu. mol) and 4-dimethylaminopyridine (6.6mg, 53.5. mu. mol) were added in this order and stirred at room temperature for 16 hours. The reaction mixture was diluted with 10mL of dichloromethane, washed with water (10 mL. times.1) and saturated brine (10 mL. times.2), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 19c (159mg, yield: 51.0%)

The fourth step

2, 4-Dimethoxybenzyl (S) -2- ((2-Aminoacetamido) methoxy) -2-cyclopropylacetate 19d

19c (60mg, 104.4. mu. mol) was dissolved in 1mL of methylene chloride, 0.5mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. Concentrating the reaction solution under reduced pressure, adding 2mL of toluene, concentrating under reduced pressure, and repeating twice; add 3mL of n-hexane for slurry, pour off the upper n-hexane layer, repeat three times, concentrate under reduced pressure to give the crude title product 19d (21mg) which was used in the next reaction without purification.

The fifth step

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-pentaazaeicosa-20-oic acid ester 19e

Crude 19d (36mg, 102.2. mu. mol) was dissolved in 4mL of N, N-dimethylformamide, 17i (52mg, 103.6. mu. mol) was added, and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (34.6mg, 125.0. mu. mol) was added and stirred at room temperature for 1 hour. 10mL of water was added, extraction was performed with ethyl acetate (10 mL. times.3), and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10 mL. times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 19e (70mg, yield: 80.2%).

The sixth step

(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-pentaazaeicosa-20-oic acid 19f

19e (70mg, 83.7. mu. mol) was dissolved in 2.5mL of a 3% (v/v) solution of dichloroacetic acid in dichloromethane, cooled to 0-5 ℃ in an ice water bath, triethylsilane (29mg, 249.4. mu. mol) was added, and the reaction was stirred in an ice bath for 3 hours. Half of the organic solvent was removed by concentration under reduced pressure in an ice bath, 5mL of diethyl ether was added, the mixture was naturally warmed to room temperature and slurried, a white solid was precipitated, filtered, and the cake was collected and drained by an oil pump to give the title product 19f (57mg, yield: 99.2%).

Seventh step

(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

Adding 1b (30mg, 63.6 mu mol) into a reaction bottle, adding 1mL of 10% (v/v) methanol dichloromethane solution, replacing with argon for three times, cooling to 0-5 ℃ in an ice water bath, adding a drop of triethylamine, and stirring until 1b is dissolved. 19f (57mg, 83.1. mu. mol) was dissolved in 1mL of a 10% (v/v) solution of methanol in methylene chloride, and then added dropwise to the above reaction solution, followed by addition of 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (26mg, 93.9. mu. mol). The reaction was warmed to room temperature and stirred for 1 hour. Adding 10mL of dichloromethane and 5mL of water, stirring for 5 minutes, standing for layering, and collecting an organic phase; the aqueous phase was extracted with dichloromethane (10 mL. times.3) and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (10 mL. times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 19g (56mg, yield: 79.8%).

MS m/z(ESI):1103.1[M+1]。

Eighth step

(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

19g (4.6mg, 4.16. mu. mol) was dissolved in 1.5mL of methylene chloride, and 0.75mL of diethylamine was added thereto and stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure, 2mL of toluene was added and concentrated under reduced pressure twice, 3mL of n-hexane was added and slurried, the upper n-hexane layer was decanted and repeated three times, and concentrated under reduced pressure to give the crude title product 19h (4.0mg) which was used in the next reaction without purification.

The ninth step

(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-hexaoxy-3, 20,23,26,29,32,35,38,41, 44-decaoxa-5, 8,11,14, 17-pentaazatetrahexadec-46-yl) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxamide 19

The crude product 19h (4.0mg) was dissolved in 1mL of N, N-dimethylformamide, 17g (2.9mg, 4.2. mu. mol) was added, and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (1.5mg, 5.4. mu. mol) was added and stirred at room temperature for 40 minutes. The reaction mixture was filtered and purified by HPLC (separation conditions: column: Xbridge Prep C18 OBD 5 μm 19 x 250 mm; mobile phase: A-water (10mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 19(2.1mg, yield: 32.4%).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.71-8.62(m,1H),8.59-8.51(m,1H),8.34-8.26(m,1H),8.14-8.02(m,2H),7.95-7.86(m,1H),7.83-7.69(m,2H),7.35-7.31(m,1H),7.29-7.11(m,3H),7.01(s,1H),6.72-6.50(m,3H),5.59-5.50(m,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)。

Examples 1 to 20 (reference example)

The title compound 20 was synthesized by reference to the procedure provided in example 58 "on page 163 of the specification in the patent" CN104755494A ".

The following antibodies can be prepared by a conventional method for antibody production, and can be obtained by, for example, vector construction, transfection of eukaryotic cells such as HEK293 cells (Life Technologies Cat. No.11625019), expression purification, and the like.

The following is the sequence of Trastuzumab:

light chain

Heavy chain

The following is the sequence of Pertuzumab:

light chains

Heavy chain

The following is the sequence of B7H3 antibody 1F9 DS:

light chains

Heavy chain

Examples 1 to 21 ADC-1

An aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.082mL, 0.82. mu. mol) was added to an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 2.5mL, 9.96mg/mL, 0.168. mu. mol) of the antibody Trastuzumab (Trastuzumab) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath oscillator and reacting the mixture at 37 ℃ for 3 hours with shaking; the reaction solution was cooled to 25 ℃ with a water bath, diluted to 5.0mg/mL, and 2.0mL of the solution was taken out and reacted.

Compound 10-short retention time Compound (2.1mg, 2.02. mu. mol) was dissolved in 0.10mL of DMSO, added to the above 2.0mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (5.0mg/mL, 1.1mL) of an exemplary product ADC-1 of formula FADC-1, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 5.09.

Examples 1-22 ADC-2

To a PBS buffer aqueous solution (0.05M PBS buffer aqueous solution with pH 6.5; 2.5mL, 9.96mg/mL, 0.168. mu. mol) of an antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.082mL, 0.82. mu. mol) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath oscillator and shaking the mixture at 37 ℃ for 3 hours; the reaction solution was cooled to 25 ℃ by a water bath, diluted to 5.0mg/mL, and 2.0mL of the solution was taken out and reacted.

Compound 10-longer retention time Compound (2.1mg, 2.02. mu. mol) was dissolved in 0.10mL of DMSO, added to the above 2.0mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (4.95mg/mL, 1.1mL) of an exemplary product ADC-2 of formula FADC-1, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 7.39.

Examples 1 to 23 ADC-3

An aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.082mL, 0.82. mu. mol) was added to an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 2.5mL, 9.96mg/mL, 0.168. mu. mol) of the antibody Trastuzumab (Trastuzumab) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath oscillator and reacting the mixture at 37 ℃ for 3 hours with shaking; the reaction solution was cooled to 25 ℃ by a water bath, diluted to 5.0mg/mL, and 2.0mL of the solution was taken out and reacted.

Compound 8(2.1mg, 2.02. mu. mol) was dissolved in 0.10mL of DMSO, added to the above 2.0mL of solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA) to give PBS buffer (5.24mg/mL, 1.1mL) of the exemplary product ADC-3 of the general formula FADC-3, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 7.36.

Examples 1-24 ADC-4

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 3.74mL, 13.38mg/mL, 0.338. mu. mol) of an antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.173mL, 1.73. mu. mol) at 37 ℃ and the mixture was placed in a water bath oscillator and reacted at 37 ℃ for 3 hours with shaking to stop the reaction; the reaction solution was cooled to 25 ℃ with a water bath, diluted to 6.7mg/mL, and 1.3mL of the solution was taken out and allowed to react.

Compound 9-short Retention time Compound 9-A (1.0mg, 0.93. mu. mol) was dissolved in 0.10mL of DMSO, added to the above 1.3mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.72mg/mL, 2.36mL) of ADC-4, an exemplary product of the general formula FADC-4A, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 7.39.

Examples 1 to 25 ADC-5

To a PBS buffer aqueous solution (0.05M PBS buffer aqueous solution with pH 6.5; 3.0mL, 6.70mg/mL, 0.136. mu. mol) of an antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.067mL, 0.67. mu. mol) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath oscillator and shaking the mixture at 37 ℃ for 3 hours; the reaction solution was cooled to 25 ℃ with a water bath, and 0.614mL of the solution was taken out and reacted downward.

Compound 9-short Retention time Compound 9-A (0.5mg, 0.42. mu. mol) was dissolved in 0.031mL of DMSO, added to the above 0.614mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA-containing) to give PBS buffer (3.08mg/mL, 0.82mL) of ADC-5, an exemplary product of the general formula FADC-4A, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 3.16.

Examples 1 to 26 ADC-6

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 3.74mL, 13.38mg/mL, 0.338. mu. mol) of an antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.173mL, 1.73. mu. mol) at 37 ℃ and the mixture was placed in a water bath oscillator and reacted at 37 ℃ for 3 hours with shaking to stop the reaction; the reaction solution was cooled to 25 ℃ with a water bath, diluted to 6.7mg/mL, and 0.75mL of the solution was taken out and allowed to react.

Compound 9-longer Retention time Compound 9-B (0.68mg, 0.63. mu. mol) was dissolved in 0.10mL of DMSO, added to the above 0.75mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was purified by desalting on a Sephadex G25 gel column (eluent: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA) to give PBS buffer (1.78mg/mL, 1.78mL) of ADC-6, an exemplary product of the general formula FADC-4B, and stored at 4 ℃.

UV-HPLC calculation mean: n is 3.94.

Examples 1 to 27 ADC-7

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 5.0mL, 10mg/mL, 0.338 μmol) of an antibody Pertuzumab (Pertuzumab) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.173mL, 1.73 μmol) was added, placed in a water bath oscillator, reacted at 37 ℃ for 3 hours with shaking, and the reaction was stopped; the reaction solution was cooled to 25 ℃ with a water bath, diluted to 5.0mg/mL, and 1.0mL of the solution was taken out and reacted.

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

UV-HPLC calculation mean: n is 6.91.

Examples 1-28 ADC-8

To a PBS buffer aqueous solution (0.05M aqueous PBS buffer solution with pH 6.5; 5.0mL, 10mg/mL, 0.338. mu. mol) of an antibody Pertuzumab (Pertuzumab) at 37 ℃ was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.173mL, 1.73. mu. mol), and the mixture was placed in a water bath oscillator and reacted at 37 ℃ for 3 hours with shaking to stop the reaction; the reaction solution was cooled to 25 ℃ by a water bath, diluted to 5.0mg/mL, and 1.6mL of the solution was taken out and reacted.

Compound 10-short retention time Compound (1.04mg, 1.0. mu. mol) was dissolved in 0.1mL of DMSO, added to the above 1.6mL of solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (2.14mg/mL, 2.31mL) of an exemplary product ADC-8 of formula FADC-8, which was stored at 4 ℃.

UV-HPLC calculation mean: n is 6.58.

Examples 1 to 29 ADC-9

To a PBS buffer aqueous solution (0.05M aqueous PBS buffer solution with pH 6.5; 5.0mL, 10mg/mL, 0.338. mu. mol) of an antibody Pertuzumab (Pertuzumab) at 37 ℃ was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.173mL, 1.73. mu. mol), and the mixture was placed in a water bath oscillator and reacted at 37 ℃ for 3 hours with shaking to stop the reaction; the reaction solution was cooled to 25 ℃ by a water bath, diluted to 5.0mg/mL, and 0.8mL of the solution was taken out and reacted.

Compound 9-short Retention time Compound 9-A (0.55mg, 0.5. mu. mol) was dissolved in 0.1mL of DMSO, added to the above 0.8mL solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA) to give PBS buffer (2.27mg/mL, 1.11mL) of ADC-9, an exemplary product of the general formula FADC-9A, and stored at 4 ℃.

UV-HPLC calculation mean: n is 3.16.

Examples 1-30 ADC-10

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.574mL, 38.78nmol) of the antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 19.76. mu.L, 197.6nmol) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.25.

Examples 1 to 31 ADC-11

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.646mL, 43.64nmol) of the antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 22.24. mu.L, 222.4nmol) at 37 ℃ and the mixture was placed in a water bath shaker and reacted for 3 hours with shaking at 37 ℃ to stop the reaction. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 14-longer retention time compound (0.72mg, 662nmol) was dissolved in 40. mu.l DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA) to give PBS buffer (2.13mg/mL, 1.87mL) of the exemplary product ADC-11 of the general formula FADC-10, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.03.

Examples 1-32 ADC-12

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 0.726mL, 49.05nmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 25.0 μ L, 250.0nmol) was added, placed in a water bath shaker, and reacted at 37 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.93.

Examples 1-33 ADC-13

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 0.287mL, 19.39nmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 9.88 μ L, 98.8nmol) was added, placed in a water bath shaker, and reacted at 37 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.53.

Examples 1-34 ADC-14

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 0.592mL, 40.0nmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 20.38 μ L, 203.8nmol) was added, placed in a water bath shaker, and reacted at 37 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 17(0.92mg, 598nmol) was dissolved in 40. mu.l DMSO, added to the above reaction mixture, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (0.30mg/mL, 12.0mL) of the exemplary product ADC-14 of formula FADC-14, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.61.

Examples 1-35 ADC-15

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 0.592mL, 40.0nmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 20.38 μ L, 203.8nmol) was added, placed in a water bath shaker, and reacted at 37 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 7.89.

Examples 1-36 ADC-16

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.53mL, 35.8nmol) of the antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 18.25. mu.L, 182.5nmol) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 19(0.83mg, 534nmol) was dissolved in 35. mu.l DMSO, and the resulting solution was added to the reaction mixture, and the reaction mixture was stirred in a water bath shaker at 25 ℃ for 3 hours to stop the reaction. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, 0.001M EDTA-containing) to give PBS buffer (0.32mg/mL, 12.0mL) of an exemplary product ADC-16 of formula FADC-16, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.43.

Examples 1-37 ADC-17

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 2.0mL, 135.12nmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 43.2 μ L, 432nmol) was added, placed in a water bath shaker, and the reaction was stopped by shaking at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 5.42.

EXAMPLES 1-38 ADC-18 (reference example)

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 1.5mL, 101.3nmol) at 37 ℃, an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 51.7 μ L, 517nmol) was added, placed in a water bath shaker, and the reaction was stopped by shaking at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 7.23.

Examples 1-39 ADC-19

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 1.36mL, 91.9nmol) of the antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 46.9. mu.L, 469nmol) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 9-shorter retention time Compound 9-A (2.0mg, 1862nmol) was dissolved in 100. mu.l DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (0.73mg/mL, 13.0mL) of ADC-19, an exemplary product of the general formula FADC-4A, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 6.26.

EXAMPLES 1-40 ADC-20

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer pH 6.5; 10.0mg/mL, 1.5mL, 101.3nmol) at 37 ℃, an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 51.7 μ L, 517nmol) was added, placed in a water bath shaker, and the reaction was stopped by shaking at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 10-longer retention time Compound (2.0mg, 1815nmol) was dissolved in 100. mu.l DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (0.73mg/mL, 13.0mL) of an exemplary product ADC-20 of formula FADC-1, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.43.

EXAMPLES 1-41 ADC-21 (reference example)

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer at pH 6.5; 10.0mg/mL, 1.86mL, 125.4nmol) at 37 ℃, an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 63.9 μ L, 639nmol) was added, and the reaction was stopped by shaking the mixture at 37 ℃ in a water bath shaker for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 7.23.

Examples 1-42 ADC-22

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 1.88mL, 127.2nmol) of the antibody Trastuzumab (Trastuzumab) was added a prepared aqueous solution (10mM, 64.9. mu.L, 649nmol) of tris (2-carboxyethyl) phosphine (TCEP) at 37 ℃ and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.79.

EXAMPLES 1-43 ADC-23 (reference example)

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer at pH 6.5; 10.0mg/mL, 345mL, 23.31 μmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 11.89mL, 118.9 μmol) was added, placed in a water bath shaker, and the reaction was stopped by shaking at 37 ℃ for 3.5 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 20(362mg, 350. mu. mol) was dissolved in 7.12mL of MeCN and 3.56mL of DMSO, added to the above reaction mixture, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was purified by desalting through an ultrafiltration membrane with a PBS buffered aqueous solution (0.05M PBS buffered aqueous solution with pH 6.5) containing 2% (v/v) MeCN and 1% (v/v) DMSO followed by a succinic acid buffered aqueous solution (0.01M succinic acid buffered aqueous solution with pH 5.3), then sucrose was added to 60mg/mL and tween 20 to 0.2mg/mL, and lyophilized to obtain a lyophilized powder sample of the exemplary product ADC-23 of general formula FADC-18, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.05.

Examples 1-44 ADC-24

To a solution of Trastuzumab (Trastuzumab) in PBS buffer (0.05M aqueous PBS buffer at pH 6.5; 10.0mg/mL, 332mL, 22.43 μmol) at 37 ℃, a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 11.44mL, 114.4 μmol) was added, placed in a water bath shaker, and the reaction was stopped by shaking at 37 ℃ for 3.5 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 9-short Retention time Compound 9-A (241mg, 224. mu. mol) was dissolved in 13.76mL of MeCN and 6.88mL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was purified by desalting through an ultrafiltration membrane with a PBS buffered aqueous solution (0.05M PBS buffered aqueous solution with pH 6.5) containing 4% (v/v) MeCN and 2% (v/v) DMSO followed by a succinic acid buffered aqueous solution (0.01M succinic acid buffered aqueous solution with pH 5.3), then sucrose was added to 60mg/mL and tween 20 to 0.2mg/mL, and lyophilized to obtain a lyophilized powder sample of the exemplary product ADC-24 of FADC-4A formula, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 7.07.

Examples 1-45 ADC-25

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 2.14mL, 144.60nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 73.7 μ L, 740nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.87.

EXAMPLES 1-46 ADC-26 (reference example)

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.89mL, 60.14nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 30.1 μ L, 300nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 20(1.0mg, 967nmol) was dissolved in 100. mu.l DMSO, added to the above reaction mixture, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.61mg/mL, 4.0mL) of an exemplary product ADC-26 of formula FADC-26, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 6.15.

Examples 1-47 ADC-27

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.89mL, 60.14nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 30.1 μ L, 300nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 9-shorter retention time Compound 9-A (1.02mg, 950nmol) was dissolved in 100. mu.l DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.94mg/mL, 3.5mL) of ADC-27, an exemplary product of the general formula FADC-25, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 6.11.

EXAMPLES 1-48 ADC-28 (reference example)

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 2.36mL, 159.47nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 81.3 μ L, 810nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 7.46.

Examples 1-49 ADC-29

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.80mL, 50.06nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 28.6 μ L, 290nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, placed in a water bath shaker, and the reaction was stopped with shaking at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 7.24.

EXAMPLES 1-50 ADC-30 (reference example)

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.86mL, 58.4nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 29.1 μ L, 290nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, placed in a water bath shaker, and the reaction was stopped with shaking at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

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

UV-Vis calculation of mean: n is 6.15.

Examples 1 to 51 ADC-31

To an aqueous PBS buffer solution (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 0.89mL, 60.14nmol) of antibody B7H3, antibody 1F9DS, a prepared aqueous solution (10mM, 30.1 μ L, 300nmol) of tris (2-carboxyethyl) phosphine (TCEP) was added at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 8(1.0mg, 943nmol) was dissolved in 100. mu.l DMSO, and the resulting solution was added to the reaction mixture, and the reaction mixture was stirred in a water bath shaker at 25 ℃ for 3 hours to stop the reaction. The reaction was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.47mg/mL, 4.5mL) of an exemplary product ADC-31 of the general formula FADC-31, which was stored at 4 ℃.

UV-Vis calculation of mean: n is 6.33.

ADC stock solution drug load analysis

Purpose and principle of experiment

ADC stock solution is an antibody cross-linked drug, and the mechanism of treating diseases is to transport toxin molecules into cells depending on the targeting of the antibody so as to kill the cells. The drug loading plays a decisive role in the drug efficacy. The drug loading of the ADC stock solution was determined using the uv method.

Experimental methods

After the cuvette filled with the sodium succinate buffer solution is respectively placed in a reference absorption cell and a sample determination absorption cell, and the solvent blank is deducted, the cuvette filled with the test solution is placed in the sample determination absorption cell, and the absorbance at 280nm and 370nm is determined.

And (4) calculating a result: the loading capacity of the ADC stock solution is measured by adopting an ultraviolet spectrophotometry (using an instrument: a Thermo nanodrop2000 ultraviolet spectrophotometer), and the principle is that the total absorbance value of the ADC stock solution under a certain wavelength is equal to the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody under the wavelength, namely:

(1)A _280nm ＝ε _mab-280 bC _mab +ε _Drug-280 bC _Drug

ε _Drug-280 : the average molar extinction coefficient of the drug at 280nm is 5100;

C _Drug : the concentration of the drug;

ε _mab-280 : the average molar extinction coefficient of the trastuzumab stock solution or the pertuzumab stock solution at 280nm is 214600;

C _mab : the concentration of trastuzumab stock solution or pertuzumab stock solution;

b: the optical path length is 1 cm.

The same equation can be obtained for the total absorbance value of the sample at 370 nm:

(2)A _370nm ＝ε _mab-370 bC _mab +ε _Drug-370 bC _Drug

ε _Drug-370 : the mean molar extinction coefficient of the drug at 370nm is 19000;

C _Drug : the concentration of the drug;

ε _mab-370 : the extinction coefficient of the trastuzumab stock solution or the pertuzumab stock solution at 370nm is 0;

C _mab : the concentration of trastuzumab stock solution;

b: the optical path length was 1 cm.

The drug loading can be calculated by combining two equations (1) and (2) with the extinction coefficient and concentration data of the monoclonal antibody and the drug at two detection wavelengths.

Drug loading ═ C _Drug /C _mab 。

Biological evaluation

Test example 1-1: in vitro tumor cell proliferation inhibition assay with compounds of the disclosure

First, test purpose

The purpose of this experiment was to examine the inhibitory activity of the pharmaceutical compounds of the present disclosure on the proliferation of U87MG cells (Chinese academy of cells, Catalog # TCTU 138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Cat. RTM. HTB-30) in vitro. Treating cells with different concentrations of compounds in vitro, culturing for 6 days, and culturing with CTG: (Luminescent Cell visual Assay, Promega, cat #: g7573) The proliferation of cells was measured by the reagents and the in vitro activity of the compound was assessed according to the IC50 value.

Second, Experimental methods

The following is an example of an in vitro proliferation inhibition assay for U87MG cells, which is illustrative of the method of the present disclosure for testing compounds of the present disclosure for in vitro proliferation inhibitory activity against tumor cells. The method is equally applicable, but not limited to, testing for proliferation inhibitory activity in vitro on other tumor cells.

1. Cell culture: u87MG and SK-BR-3 cells were cultured in EMEM medium (GE, cat # SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, cat # 16600-108) containing 10% FBS, respectively.

2. Cell preparation: taking U87MG and SK-BR-3 cells in logarithmic phase, washing with PBS (phosphate buffer solution, Shanghai culture Biotech Co., Ltd.) for 1 time, adding 2-3mL of Trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies Co., Ltd.) to digest for 2-3min, adding 10-15mL of cell culture solution after the cells are completely digested, eluting the digested cells, centrifuging at 1000rpm for 5min, discarding the supernatant, then adding 10-20mL of cell culture solution to resuspend the cells, and preparing into single cell suspension.

3. Cell plating: mixing U87MG and SK-BR-3 single cell suspension, and adjusting viable cell density to 2.75 × 10 with cell culture medium ³ cells/mL and 8.25X 10 ³ cells/mL, the cell suspension after density adjustment was mixed well and added to a 96-well cell culture plate at 180. mu.L/well. Only 200. mu.L of the medium was added to the peripheral wells of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃, 5% CO) ₂ )。

4. Preparation of compound: the compound was dissolved in DMSO (dimethylsulfoxide, Shanghai Tatankojic Co., Ltd.) to prepare a stock solution having an initial concentration of 10 mM.

The initial concentration of the small molecule compound was 500nM, as follows.

Respectively adding 30 mu L of different samples to be detected into a first column of a 96-hole U-shaped bottom dispensing plate, wherein the concentration of the samples is 100 mu M; add 20. mu.L DMSO to each well from column 2 to column 11. Take 10. mu.L of the first column sample into the second column 20. mu.L DMSO, mix well, take 10. mu.L into the third column, and so on to column 10. And taking 5 mu L of the medicines in each hole of the medicine preparation plate to 95 mu L of EMEM culture medium, and uniformly mixing for later use.

The starting concentration of ADC was 10nM or 500nM, as dosed below.

Respectively adding 100 mu L of different samples to be detected in a first column of a 96-well plate, wherein the concentration of the samples is 100nM or 5 mu M; column 2 to column 11 each well was filled with 100. mu.L of PBS. Take 50 μ L of the first column sample into the second column 100 μ L PBS, mix well, take 50 μ L into the third column, and so on to dilute 3 times into the 10 th column.

5. Sample adding operation: to the plate 20 μ L of each sample was added in duplicate wells at different concentrations. The plates were incubated in an incubator for 6 days (37 ℃, 5% CO) ₂ )。

6. And (3) color development operation: the 96-well cell culture plate was removed, 90. mu.L of CTG solution was added to each well, and incubated at room temperature for 10 minutes.

7. And (3) plate reading operation: the 96-well cell culture plate was removed and placed in a microplate reader (BMG labtech, PHERAstar FS) and chemiluminescence was measured with the microplate reader.

Third, data analysis

Data were processed and analyzed using Microsoft Excel, Graphpad Prism 5. See table 1 below for experimental results.

TABLE 1 IC inhibition of in vitro proliferation of SK-BR-3 cells and U87 cells by small molecule fragments of the present disclosure ₅₀ Value of

And (4) conclusion: the small molecule fragment in the disclosure has obvious proliferation inhibition activity on SK-BR-3 cells and U87 cells, and the chiral center has certain influence on the inhibition activity of the compound.

Test examples 1 to 2: in vitro proliferation inhibition assay for HER2 targeted tumor cells by antibody drug conjugates of the disclosure

The purpose of this experiment was to detect antibody drug conjugates of the present disclosure against the HER2 target in vitro on SK-BR-3 (human breast cancer cells, ATCC, Cat. HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, Cat. HTB-132)Inhibitory activity of proliferation. Treating cells with different concentrations of compounds in vitro, culturing for 6 days, detecting cell proliferation with CTG reagent according to IC ₅₀ Values the in vitro activity of the compound was evaluated.

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 10% FBS-containing McCoy's 5A medium (Gibco, cat # 16600-108), 10% FBS-containing EMEM medium (GE, cat # SH30024.01), and 10% FBS-containing L-15 medium (ThermoFisher, cat # 11415-114), respectively. Adjusting the viable cell density of the three cells to 8.33 × 10 respectively by using cell culture solution ³ Individual cell/mL, 8.33X 10 ³ Individual cells/mL and 1.39X 10 ⁴ The cell suspension after density adjustment was mixed well per cell/mL, and added to a 96-well cell culture plate at 180. mu.L/well. The results obtained by testing the related compounds are shown in Table 2 below.

TABLE 2 IC of in vitro proliferation inhibition of HER2 targeted tumor cells by antibody drug conjugates of the disclosure ₅₀ Value of

And (4) conclusion: the antibody drug conjugate aiming at HER2 target has obvious proliferation inhibition activity on HER2 positive cell SK-BR-3; meanwhile, the compounds have weak proliferation inhibition activity on HER2 negative cells MDA-MB-468; has good selectivity.

Test examples 1 to 3: her2-ADC plasma stability experiment

ADC-19, ADC-18, ADC-20, human plasma, monkey plasma (Shanghai Medicy biomedical Co., Ltd.), and 1% BSA (Sigma) PBS solution (Shanghai Biotech) were each sterilized by filtration through a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the sterile plasma or 1% BSA in PBS, respectively, at a final concentration of 200. mu.g/mL, incubated at 37 ℃ in a cell incubator, the day of incubation was noted as day 0, and samples were removed on days 7, 14, and 21, respectively, for detection of free toxin.

Taking 25 mu L of sample to a 96-well plate; adding 50 μ L of internal standard working solution (100ng/mL camptothecin acetonitrile solution) and 150 μ L acetonitrile; vortex for 5 minutes, centrifuge for 10 minutes (4000rpm), and 5 μ L for LC/MS/MS (applied biosystems, USA) analysis.

The results show that: ADC-19 was fairly stable in both human and monkey plasma, as well as 1% BSA in PBS, with a release rate of free toxin of no more than 2.1% at the highest, and stable on day 14, see figure 1A.

ADC-18 was poorly stable in human and monkey plasma with free toxin release rates of up to 14.5% and 8.10%, respectively. The stability was compared in 1% BSA in PBS, as shown in FIG. 1B.

ADC-20 was less stable in human plasma, monkey plasma, and 1% BSA in PBS, with free toxin release rates of up to 21.7%, 29.7%, and 21.7%, respectively. And is always in a degraded state in a 1% BSAPBS solution, as shown in FIG. 1C.

Test examples 1 to 4: JIMT-1 tumor-bearing mouse efficacy evaluation

1. Purpose of the test

Nunuu nude mice are used as test animals, and the curative effect of Her2-ADC antibodies T-DM1, ADC-21 and ADC-24 on human breast cancer cells Trastuzumab (Trastuzumab) drug-resistant strain (herceptin) JIMT-1 transplantation tumor nude mice after intraperitoneal injection and administration is evaluated.

2. Test drugs and materials

2-1, test drugs

T-DM1 (made by reference US 20050169933)

ADC-21：3mg/kg

ADC-21：10mg/kg

ADC-24：3mg/kg

ADC-24：10mg/kg

Blank control (Blank): PBS (Poly Butylene succinate)

2-2, a preparation method: all are diluted with PBS.

2-3, test animals

nude mice, purchased from Beijing vitamin Tonglihua.

3. Test method

In the mouse right costal skinInoculated with JIMT-1 cells (Nanjing Kebai) (5X 10) ⁶ /only, with 50% artificial basement membrane), tumors grew 8 days, growing to 203.09 ± 11.94mm ³ The animals were then randomly assigned (d1) 8 animals per group for 6 groups.

The medicine is administered by intraperitoneal injection for 2 times. Tumor volume and body weight were measured 2 times per week and data were recorded.

Data statistics Excel 2003 statistical software was used: the average is calculated as avg; the SD value is calculated as STDEV; SEM values were calculated as STDEV/SQRT; p-values for differences between groups were calculated as TTEST.

Tumor volume (V) was calculated as: v1/2 xl _{Long and long} ×L _{Short length} ²

Relative volume (RTV) V _T /V ₀

Tumor inhibition rate (%) (C) _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively. C _RTV 、T _RTV Relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

4. Test results

The results of the experiment are shown in FIG. 2, and the experiment was terminated by observing the day 34 after 2 intraperitoneal injections. T-DM1(10mg/kg) has no inhibition effect on tumors; ADC-21, the tumor inhibition rate of 3mg/kg is 46.22% (P is less than 0.01); ADC-21, the tumor inhibition rate of 10mg/kg is 56.77% (P is less than 0.001); ADC-24, the tumor inhibition rate of 3mg/kg is 62.77% (P is less than 0.001); ADC-24, the tumor inhibition rate of 10mg/kg is 76.32% (P is less than 0.001). Under the condition of the same dosage, the tumor inhibition effect of the ADC-24 is obviously better than that of the ADC-21.

Test examples 1 to 5: evaluation of drug effect of SK-BR-3 tumor-bearing mice

1. Purpose of the experiment

Nuu nude mice are used as test animals to evaluate the curative effect of Her2-ADC antibodies ADC-21 and ADC-22 on human breast cancer cell SK-BR-3 transplantation tumor nude mice after intraperitoneal injection administration.

2. Test drugs and materials

2-1, test drugs

ADC-21：1mg/kg

ADC-21：6mg/kg

ADC-22：1mg/kg

ADC-22：6mg/kg

Blank control (Blank): PBS.

2-2, a preparation method: all were diluted with PBS.

2-3, test animals

nude mice, purchased from Beijing vitamin D.

3. Test method

Mice were inoculated subcutaneously with SK-BR-3 cells (ATCC) (5X 10) ⁶ /only, with 50% artificial basement membrane), tumors grew for 20 days, growing to 153.34 ± 11.73mm ³ The animals were then randomly assigned (d0) to 8 animals per group for a total of 5 groups.

The preparation is administered by intraperitoneal injection for 1 time. Tumor volumes and body weights were measured 2 times per week and data were recorded.

Data statistics Excel 2003 statistical software was used: the average is calculated as avg; the SD value was calculated as STDEV; SEM values were calculated as STDEV/SQRT; the P-value of the difference between groups was calculated as TTEST.

Tumor volume (V) was calculated as: v is 1/2 XL _{Long and long} ×L _{Short length} ²

Relative volume (RTV) V _T /V ₀

Tumor inhibition rate (%) ═ C _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively. C _RTV 、T _RTV Relative tumor volumes of the blank control and experimental groups at the end of the experiment, respectively.

4. Test results

The experimental result is shown in figure 3, the intraperitoneal injection is carried out for 1 time, the experiment is observed to be finished at the 28 th day, and the tumor inhibition rate of ADC-211 mg/kg is 15.01 percent; the tumor inhibition rate of ADC-216 mg/kg is 77.4%, and the difference is very significant compared with a blank control (P < 0.001). The tumor inhibition rate of ADC-221 mg/kg is 19.82%; the tumor inhibition rate of ADC-226 mg/kg is 98.38% (P is less than 0.001). And the tumor inhibition effect of ADC-22 is also obviously better than that of ADC-21 under the condition of 6mg/kg dose.

Test examples 1 to 6: plasma stability

Sample ADC-25 was mixed uniformly with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100. mu.g/mL, filter sterilized, incubated in a 37 ℃ water bath, day 0 was recorded on the day of incubation, and samples were removed on days 7, 14, and 21, respectively, and free toxin was detected.

Taking out samples at different time points, then placing the samples to room temperature, and uniformly mixing the samples in a vortex manner; taking 25 mu L of sample to a 96-well plate; adding 50 μ L of internal standard working solution (100ng/mL camptothecin acetonitrile solution) and 150 μ L acetonitrile; vortex for 5 minutes, centrifuge for 10 minutes (4000rpm), and take 5. mu.L of supernatant for LC/MS/MS analysis.

The results are shown in figure 4, ADC-25 is fairly stable in both human and monkey plasma, as well as in 1% BSA PBS, with a release rate of free toxin of no more than 2% at the highest, and tends to be stable on day 14.

Test examples 1 to 7: evaluation of therapeutic Effect of ADC on human brain astrocytoma U87MG nude mouse transplantation tumor

1. Purpose of the experiment

In this experiment, BALB/cA-nude mice were used as test animals to evaluate the efficacy of the ADC compounds of the present disclosure on human brain astrocytoma U87MG nude mouse transplantable tumors.

2. Test drugs and materials

2-1, test drugs

ADC-27(3mg/kg)

ADC-26(3mg/kg)

Blank control (Blank): PBS buffer, pH 7.4.

2-2, a preparation method: PBS buffer, pH 7.4.

2-3, test animals

BALB/cA-nude mice: purchased from jiesijie laboratory animals llc, shanghai.

3. Test method

BALB/cA-nude mice, female, were used for the experiment, and human brain astrocytoma U87MG cells (human brain astrocytoma, cell bank of Chinese academy, Catalog # TCTU 138) were inoculated subcutaneously for 6-7 weeks. On the tenth day after inoculation of the cells, the animals were randomly grouped (D0), 8 animals per group, administered by i.p. injection 1/week for 3 times in total, and tumor volume and body weight were measured 2-3 times per week and the data were recorded. Tumor volume (V) was calculated as:

V＝1/2×a×b ²

wherein: a. b represents length and width, respectively.

Relative volume (RTV) V _T /V ₀

Tumor inhibition rate (%) (C) _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively. C _RTV 、T _RTV Relative tumor volumes of control (blank) and experimental groups, respectively, at the end of the experiment.

4. Test results

Intraperitoneal (i.p.) administration is given 1 time per week for 3 times, and the tumor inhibition rate of ADC-273 mg/kg reaches 63.3% (P <0.0001) observed on day 22; the inhibition rate of ADC-263 mg/kg reaches 49.1%. ADC-27 showed a stronger antitumor effect than ADC-26.

The weight of each group of animals is normal in the administration process, which indicates that ADC has no obvious toxic or side effect. The results of the measurements are shown in Table 3 and FIG. 5. The detected antibody can effectively inhibit the growth of U87MG transplanted tumor in tumor-bearing nude mice and shows dose dependence.

TABLE 3 therapeutic efficacy of the administered antibody against human brain astrocytoma U87MG nude mouse graft tumor (D22)

Denotes P <0.001

Test examples 1 to 8: evaluation of curative effect of ADC on human pharyngeal cancer hydrothorax metastatic cell Detroit 562 nude mouse transplantation tumor

1. Purpose of the experiment

In the experiment, BALB/cA-nude mice are taken as test animals, and the curative effect of the ADC compound disclosed by the invention on transplanted tumor of human pharyngeal cancer hydrothorax metastatic cells Detroit 562 nude mice is evaluated.

2. Test drugs and materials

2-1, test drugs

ADC-29(3mg/kg)

ADC-28(3mg/kg)

Negative control ADC (3 mg/kg): antibody drug conjugates formed by coupling non-B7H 3 target antibodies to compound 20.

2-2, a preparation method: all were diluted with PBS.

2-3, test animals

BALB/cA-nude mice: purchased from Changzhou Kavens laboratory animals, Inc.

3. Test method

BALB/cA-nude mice for experiment, female, 6-7 weeks, are inoculated with Detroit 562 cells (ATCC, Catalog) of human pharyngeal cancer pleural effusion transfer cells subcutaneouslyCCL-138 ^TM ). On the tenth day after inoculation of the cells, the animals were randomly grouped (D0), 8 animals per group, administered initially intraperitoneally 1/week for a total of 3 times, and tumor volume and body weight were measured 2-3 times per week and data were recorded. Tumor volume (V) was calculated as:

V＝1/2×a×b ²

wherein: a. b represents length and width, respectively.

Relative volume (RTV) V _T /V ₀

Tumor inhibition rate (%) (C) _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively.C _RTV 、T _RTV The relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.

4. Test results

Intraperitoneal injection is given 1 time per week for 3 times, and the tumor inhibition rates of the tested ADC on the 28 th day are respectively observed as follows: the tumor inhibition rate of ADC-293 mg/kg (3mpk) reaches 72.27% (P < 0.001); the tumor inhibition rate of ADC-283 mg/kg (3mpk) reaches 56.2% (P < 0.001). ADC-29 all showed stronger antitumor efficacy than ADC-28.

The weights of all animals are normal in the administration process, and the ADC has no obvious toxic or side effect. The results of the measurements are shown in Table 4 and FIG. 6. The detected antibody can effectively inhibit the growth of Detroit 562 transplanted tumor in tumor-bearing nude mice, and presents dose dependency.

TABLE 4 therapeutic effect of the administered antibody on Detroit 562 transplantable tumor in nude mice bearing tumor (D28)

Denotes P <0.001

Test examples 1 to 9: drug effect evaluation of U87-MG tumor-bearing mice

1. Purpose of the experiment

Balb/c nude mice are used as test animals, and the curative effect of the B7H 3-antibody drug conjugate after intraperitoneal injection is evaluated on a human glioma cell U87MG transplantation tumor model.

2. Test drugs and materials

2-1, test drugs

ADC-30 1mg/kg

ADC-30 3mg/kg

ADC-31 1mg/kg

ADC-31 3mg/kg

Blank control (Blank): PBS (Poly Butylene succinate)

2-2, a preparation method: all were diluted with PBS.

2-3, test animals

BALB/cA-nude mice: purchased from shanghai slake laboratory animals llc.

3. Test method

Mice were inoculated subcutaneously in the right flank with U87MG cells (human brain astrocytoma, Zhongke academy of cells, Catalog # TChu138) (2.5X 10) ⁶ One), tumors grew for 14 days to 167.49mm ³ The animals were then randomly assigned (d1) to 8 animals per group for a total of 5 groups.

The preparation is administered by intraperitoneal injection for 1 time/week and 3 times. Tumor volumes and body weights were measured 2 times per week and data were recorded.

Data statistics Excel 2003 statistical software was used: the average is calculated as avg; the SD value was calculated as STDEV; SEM values were calculated as STDEV/SQRT; the P-value of the difference between groups was calculated as TTEST.

Tumor volume (V) was calculated as: v is 1/2 XL _{Long and long} ×L _{Short length} ²

Relative volume (RTV) ═ V _T /V ₀

Tumor inhibition rate (%) ═ C _RTV -T _RTV )/C _RTV (％)

Wherein V ₀ 、V _T Tumor volumes at the beginning and end of the experiment, respectively. C _RTV 、T _RTV Relative tumor volumes were obtained for the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively.

4. Test results

The experimental results are shown in fig. 7, i.e. 1 time/week of intraperitoneal injection and 3 times of total administration, and the tumor inhibition rates of the tested ADCs are respectively observed at day 18: the tumor inhibition rate of ADC-301 mg/kg is 0.31%; the tumor inhibition rate of ADC-303 mg/kg reaches 45.23% (P < 0.0001); the tumor inhibition rate of ADC-311 mg/kg reaches 39.22% (P < 0.01); the tumor inhibition rate of ADC-313 mg/kg reaches 80.24% (P < 0.0001). Under the condition of the same dosage, the tumor inhibition effect of the ADC-31 is obviously better than that of the ADC-30.

Secondly, optimizing the preparation process

Exemplary products of the following examples have the structure shown below:

example 2-1

An antibody stock solution (0.0004251mmol, prepared by diluting Trituzumab antibody to a final antibody concentration of 15mg/mL with 20mM histidine-HCl buffer solution) containing 61.71mg of trastuzumab was reacted with 0.7311mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.002550mmol) in 20mM histidine-HCl buffer solution (pH 5.6) containing 2.5mM EDTA under stirring in a constant temperature water bath at 0 ℃ for 3 hours to give an intermediate I solution.

Compound 9, Compound 9-A (3.196mg, 0.002975mmol) was dissolved in 0.2062mL of DMSO to generate a DMSO solution of compound 9-A. 0.2052mL of DMSO was added to the above solution of intermediate I, and the above solution of compound 9-A in DMSO was added to the pre-DMSO solution of intermediate I, and the reaction was stirred in a water bath at 25 ℃ for 1 hour and quenched by addition of excess cysteine. An exemplary product ADC-2-1 of the general formula FADC-4A was obtained.

Mean values were calculated by reverse phase chromatography-mass spectrometry: n is 5.45.

Examples 2 to 2

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

Compound 9, Compound 9-A (3.196mg, 0.002975mmol) was dissolved in 0.2062mL of DMSO to generate a DMSO solution of compound 9-A. 0.2052mL of DMSO was added to the above solution of intermediate I, and the above solution of compound 9-A in DMSO was added to the pre-DMSO solution of intermediate I, and the reaction was stirred in a water bath at 25 ℃ for 1 hour and quenched by addition of excess cysteine. An exemplary product ADC-2-2 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.49.

Examples 2 to 3

An antibody stock solution (0.0004251 mmol) containing 61.71mg of trastuzumab (the antibody of the trastuzumab was diluted with 20mM histidine-hydrochloric acid buffer solution to a final antibody concentration of 15mg/mL) was reacted with 0.4021mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.001403mmol) in 20mM histidine-hydrochloric acid buffer solution (pH 5.6) containing 2.5mM EDTA at 25 ℃ for 3 hours with stirring in a thermostatic water bath to give a solution of intermediate I.

Compound 9, Compound 9-A (3.196mg, 0.002975mmol) was dissolved in 0.2062mL of DMSO to generate a DMSO solution of compound 9-A. 0.2052mL of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added, and the reaction was stirred in a water bath at 25 ℃ for 1 hour, and quenched by adding excess cysteine. An exemplary product ADC-2-3 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.39.

Examples 2 to 4

An antibody stock solution (0.0004251 mmol) containing 61.71mg of trastuzumab (the antibody of the trastuzumab was diluted with 20mM histidine-hydrochloric acid buffer solution to a final antibody concentration of 15mg/mL) was reacted with 0.3899mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.001360mmol) in 20mM histidine-hydrochloric acid buffer solution (pH 5.6) containing 2.5mM EDTA at 28 ℃ for 3 hours with stirring in a thermostatic water bath to give a solution of intermediate I.

Compound 9, Compound 9-A (3.196mg, 0.002975mmol) was dissolved in 0.2062mL of DMSO to generate a DMSO solution of compound 9-A. 0.2052mL of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added, and the reaction was stirred in a water bath at 25 ℃ for 1 hour, and quenched by adding excess cysteine. An exemplary product ADC-2-4 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.44.

Examples 2 to 5

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

Compound 9, Compound 9-A (3.196mg, 0.002975mmol) was dissolved in 0.2062mL of DMSO to generate a DMSO solution of compound 9-A. 0.2052mL of DMSO was added to the above solution of intermediate I, and the above solution of compound 9-A in DMSO was added to the pre-DMSO solution of intermediate I, and the reaction was stirred in a water bath at 25 ℃ for 1 hour and quenched by addition of excess cysteine. An exemplary product ADC-2-5 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.46.

Examples 2 to 6

An antibody stock solution (0.0003790 mmol) containing 55.02mg of trastuzumab (diluting the trastuzumab antibody with 50mM PBS buffer to a final antibody concentration of 15mg/mL) was reacted with 0.4563mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.001592mmol) in 50mM PBS buffer (pH 6.5) containing 2.5mM EDTA at 13 ℃ with stirring in a thermostatic water bath for 3 hours to give an intermediate I solution.

Compound 9, Compound 9-A (2.850mg, 0.002653mmol) with short retention time was dissolved in 0.1839mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829mL of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added, and the reaction was stirred in a water bath at 25 ℃ for 1 hour, and quenched by adding excess cysteine. An exemplary product ADC-2-6 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.39.

Examples 2 to 7

An antibody stock solution (0.0003790 mmol) containing 55.02mg of trastuzumab (the antibody was diluted with 50mM PBS buffer to a final antibody concentration of 15mg/mL) was reacted with 0.3477mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.001213mmol) in 50mM PBS buffer (pH 6.5) containing 2.5mM EDTA under stirring in a thermostatic water bath at 25 ℃ for 3 hours to give a solution of intermediate I.

Compound 9, Compound 9-A (2.850mg, 0.002653mmol) with short retention time was dissolved in 0.1839mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829mL of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added, and the reaction was stirred in a water bath at 25 ℃ for 1 hour, and quenched by adding excess cysteine. An exemplary product ADC-2-7 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.50.

Examples 2 to 8

An antibody stock solution (0.0003790 mmol) containing 55.02mg of trastuzumab (the antibody was diluted with 50mM PBS buffer to a final antibody concentration of 15mg/mL) was reacted with 0.3259mg of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 0.001137mmol) in 50mM PBS buffer (pH 6.5) containing 2.5mM EDTA under stirring in a thermostatic water bath at 37 ℃ for 3 hours to give a solution of intermediate I.

Compound 9, Compound 9-A (2.850mg, 0.002653mmol) with short retention time was dissolved in 0.1839mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829mL of DMSO was added to the above intermediate I solution, and the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added, and the reaction was stirred in a water bath at 25 ℃ for 1 hour, and quenched by adding excess cysteine. An exemplary product ADC-2-8 of the general formula FADC-4A was obtained.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.47.

Examples 2 to 9

127.4g of a stock antibody solution containing trastuzumab (0.88mmol, diluted with 20mM histidine-HCl buffer to a final antibody concentration of 15mg/mL) was reacted with 0.83g of tris (2-carboxyethyl) phosphine hydrochloride (Sigma, 2.90mmol) in 20mM histidine-HCl buffer (pH 5.6) containing 2.5mM EDTA in a thermostatic water bath with stirring at 25 ℃ for 3 hours to give a solution of intermediate I.

Compound 9, Compound 9-A (6.6g, 6.14mmol) was dissolved in 0.43L DMSO to produce a DMSO solution of compound 9-A. 0.43L of DMSO was added to the above intermediate I solution, and then the above DMSO solution of compound 9-A was added to the intermediate I solution in which DMSO was added in advance, and the reaction was stirred in a water bath at 25 ℃ for 1 hour to stop the reaction.

The reaction solution was purified by Capto S Impact (GE) cation chromatography column, washed with not less than 9 column volumes of 0.05M acetate buffer (pH 5.5) containing 10% (v/v) DMSO and 6 column volumes of 0.05M acetate buffer (pH 5.5), and eluted with 0.05M acetate buffer (pH 5.5, containing 0.39M sodium chloride) to remove free toxins and residual solvent from the reaction solution. The cation eluate was subjected to 7 volumes of equal volume ultrafiltration (30kd ultrafiltration membrane package) to a 0.01M succinic acid buffer (pH 5.0) at 25 ℃ to give an exemplary product ADC-A1 of formula FADC-4A. Samples of 4 batches were prepared using the method described above.

The drug loading of the 4 batches of samples is 5.7 determined by reversed phase chromatography-mass spectrometry. The four batches yield 100.8%, 98.9%, 97.4% and 99.0%, respectively.

Examples 2 to 10

To trastuzumab-containing PBS buffer (0.05M aqueous PBS buffer solution with pH 6.5; 10.0mg/mL, 164mL, 11.08 μmol) was added a prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10mM, 3.55mL, 35.5 μmol) at 37 ℃, and the reaction was stopped by placing the mixture in a water bath shaker and shaking the mixture at 37 ℃ for 3.5 hours. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 9-A (185mg, 172. mu. mol) was dissolved in 3.88mL of acetonitrile and 1.94mL of DMSO, and the resulting solution was added to the reaction mixture, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was desalted and purified by an ultrafiltration membrane using a PBS buffer aqueous solution (0.05M PBS buffer aqueous solution at pH 6.5) containing 2% (v/v) acetonitrile and 1% (v/v) DMSO followed by a succinic acid buffer aqueous solution (0.01M succinic acid buffer aqueous solution at pH 5.3) to remove small molecules, to obtain a sample of ADC-B14, an exemplary product of the general formula FADC-4A, and stored at 4 ℃.

Mean values calculated by reverse phase chromatography-mass spectrometry: n is 5.3.

Test example 2-1 drug load distribution test

RP-DAR measuring method

1.1. RP-DAR analysis was performed under the following measurement conditions:

the UPLC system comprises: waters H-Class ultra-high performance liquid chromatograph UPLC system

A detector: TUV Detector (measuring wavelength: 280nm)

A chromatographic column: waters ACQUITY UPLC Protein BEH C4(2.1 mm. times.150 mm,1.7 μm)

Column temperature: 80 deg.C

Flow rate: 0.3mL/min

Temperature of the sample chamber: 20 deg.C

Operating time: 25min

A mobile phase A: 0.1% aqueous Difluoroacetic acid (DFA)

And (3) mobile phase B: 0.1% DFA acetonitrile solution

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

Amount of sample injected: 1.0 μ L

1.2. Data analysis

Light chain of antibody when not bound to drug (L) ₀ ) And heavy chain (H) ₀ ) In contrast, when drug-bound light chain (one drug-bound light chain: l is a radical of an alcohol ₁ ) And drug-binding heavy chain (one drug-binding heavy chain: h ₁ Two drug-binding heavy chains: h ₂ Three drug-binding heavy chains: h ₃ Four drug-binding heavy chains: h ₄ ) In the case of (2), hydrophobicity increases in proportion to the number of bound drugs, and the retention time is prolonged. Thus, with L ₀ 、L ₁ 、H ₀ 、H ₁ 、H ₂ 、H ₃ And H ₄ Elution is carried out in the order of (1).

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

light chain (Epsilon LC-280)/(Epsilon LC-280+ number of conjugated drugs. times. Epsilon drug-280)

Heavy chain (Epsilon HC-280)/(Epsilon HC-280+ number of linked drugs XEpsilon drug-280)

Remarking: ε LC-280: molar extinction coefficient of light chain at 280 nm;

ε HC-28: the molar extinction coefficient of the heavy chain at 280 nm;

epsilon drug-280: molar extinction coefficient of toxin at 280 nm.

TABLE 5 reverse phase chromatogram drug loading calculation table

Name (R)	Number of connected drugs	Corrected peak area percentage
L 0	0	100 XL 0 corrected peak area/LC corrected peak area sum
L 1	1	100 XL 1 correction peak area/LC correction peak area sum
H 0	0	100 XH 0 correction peak area/HC correction peak area sum
H 1	1	100 XH 1 correction peak area/HC correction peak area sum
H 2	2	100 XH 2 correction peak area/HC correction peak area sum
H 3	3	100 XH 3 corrected peak area/HC corrected peak area sum
H 4	4	100 XH 4 correction peak area/HC correction peak area sum

Remarking: LC correction peak area sum L ₀ Corrected peak area + L ₁ Correction peak area

Total peak area corrected for HC ═ H ₀ Corrected peak area + H ₁ Corrected peak area + H ₂ Corrected peak area + H ₃ Corrected peak area + H ₄ Corrected peak area

The drug loading n is 2 × Σ (number of linked drugs × percentage of corrected peak area)/100

2. Measurement results

TABLE 6 determination of drug load distribution

Note: in ADC-2-1 to ADC-2-8, H ₄ The content of (A) is lower than the detection limit (<<1％)

The result shows that for samples prepared by adopting the same buffer system and different reduction reaction temperatures, the uniform degree of the drug loading distribution of the samples is obviously improved along with the reduction reaction temperature; for samples prepared by different buffer systems at the same reduction reaction temperature, the drug loading distribution of the samples adopting the histidine-hydrochloric acid buffer system is more uniform.

Test examples 2 to 2: free toxin assay

1. Method for measuring free toxin

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

HPLC system: waters H-Class ultra-high performance liquid chromatograph UPLC system

A detector: TUV Detector (measuring wavelength: 370nm)

A chromatographic column: waters ACQUITY UPLC Petide BEH C18( 2.1mm×150mm,1.7μm)

Column temperature: 40 deg.C

Flow rate: 0.3mL/min

Temperature of the sample chamber: 10 deg.C

A mobile phase A: 0.1% trifluoroacetic acid (TFA) in water

And (3) mobile phase B: 0.1% TFA acetonitrile solution

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

Amount of sample injected: 5.0 μ L

1.2. Data analysis

The LOD limit of the toxin is calculated as follows:

toxin limit (ppm) ═ 0.1X 4X 1000/C

Note:

a)0.1 is the toxin LOD solution concentration (. mu.g/mL), 4 is the dilution factor at the time of sample pretreatment, 1000 is the unit conversion factor, and C is the protein concentration (mg/mlmL) of the sample to be measured.

b) If the peak area of the free toxin in the sample is smaller than the peak area of the LOD solution, it is judged to be less than the limit or not detected.

2. Measurement results

Free toxin detection was performed on ADC-a1 (batches 1-4) and ADC-B14, and the results (see table 7) indicated that cation column chromatography was not only suitable for large-scale preparation, but also significantly reduced free toxin.

TABLE 7 free toxin assay

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

Claims (15)

1. A method for preparing antibody drug conjugate, wherein the structure of the antibody drug conjugate is shown as the general formula (Pc-L) _a -Y-D) is as follows:

   wherein:

   w is selected from C _1-8 Alkyl radical, C _1-8 alkyl-C _3-7 Cycloalkyl or a linear heteroalkyl of 1 to 8 atoms, said linear heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C is _1-8 Alkyl radical, C _3-7 Cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Cycloalkyl substituted with one or more substituents;

   L ² is selected from-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-、-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-、-S(CH ₂ )p ¹ C (O) -and a chemical bond, wherein p ¹ Is an integer from 1 to 20;

   L ³ is a peptide residue consisting of 2 to 7 amino acid residues, wherein said amino acid residue is selected from the group consisting of amino acid residues consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Substituted with one or more substituents in the cycloalkyl;

   R ¹ is halo C _1-6 Alkyl or C _3-7 A cycloalkyl group;

   R ² selected from hydrogen atoms, halogeno-C _1-6 Alkyl and C _3-7 A cycloalkyl group;

   or, R ¹ And R ² Together with the carbon atom to which they are attached form C _3-7 A cycloalkyl group;

   R ⁵ Selected from hydrogen atoms, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

   R ⁶ and R ⁷ Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

   m is 0 or 1;

   n is 3 to 8, n is a decimal or an integer;

   pc is an antibody or antigen-binding fragment thereof;

   the preparation method comprises the following steps:

   a step (a): reacting the antibody or antigen-binding fragment thereof with a reducing agent under reaction temperature conditions of about 1 ℃ to about 36 ℃;

   a step (b): reacting the product of step (a) with a compound represented by the following formula (La-Y-D);

   wherein: w, L ² ，L ³ ，R ¹ ，R ² ，R ⁵ ，R ⁶ ，R ⁷ And m is as defined above.
2. The method of preparing an antibody drug conjugate according to claim 1, wherein the reaction temperature conditions in step (a) are about 4 ℃ to about 30 ℃, preferably about 20 ℃ to about 30 ℃, more preferably about 25 ℃.
3. The method for preparing an antibody drug conjugate according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of about 4.5 to about 6.5; preferably, the reaction is carried out at a pH of from about 5.0 to about 6.0; more preferably, the reaction is carried out at a pH of about 5.6.
4. The method for preparing an antibody drug conjugate according to any one of claims 1 to 3, wherein the reaction in step (a) is carried out in a buffer; preferably, said buffer is selected from the group consisting of histidine salt buffers, phosphate buffers and acetate buffers; more preferably, the buffer is a histidine salt buffer containing EDTA.
5. The method for preparing an antibody drug conjugate according to any one of claims 1 to 4, wherein the reducing agent in step (a) is selected from tris (2-carboxyethyl) phosphine or a salt thereof, 1, 4-dimercaptothreitol, and β -mercaptoethanol, preferably tris (2-carboxyethyl) phosphine hydrochloride.
6. The method for preparing an antibody-drug conjugate according to any one of claims 1 to 5, wherein the method further comprises the step (c) of purifying the product of step (b) by cation column chromatography or affinity column chromatography; preferably, step (c) comprises subjecting the product of step (b) to cation column chromatography using a packing selected from Capto S Impact and Poros XS, preferably Capto S Impact.
7. The method for preparing an antibody drug conjugate according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of an anti-HER 2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Criptto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-PSMA antibody, an anti-Tenascin-Tenasc antibody, an anti-SLC 4 antibody, and an anti-Mellin binding fragment thereof;

   Preferably, the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, enobiltituzumab, Emibetuzumab, Inotuzumab, pintuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and glemtuzumab, or an antigen-binding fragment thereof.
8. The method for preparing an antibody drug conjugate according to any one of claims 1 to 7, wherein the antibody drug conjugate has a structure represented by the following formula:

   wherein n is 4 to 8, and n is a decimal or an integer.
9. The method of preparing an antibody drug conjugate according to any one of claims 1 to 8, wherein n is a decimal or integer from 4 to 8, preferably a decimal or integer from 5 to 7, more preferably a decimal or integer from 5.3 to 6.1.
10. The method of preparing an antibody drug conjugate according to any one of claims 1 to 9, wherein the drug load profile of the antibody drug conjugate is: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less;

    preferably, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and the proportion of antibody light chains in the population of antibody light chains that bind 1 drug is 65% or more.
11. A method of preparing an antibody drug conjugate, wherein the antibody drug conjugate has a structure represented by the following formula:

    wherein n is 4 to 8, and n is a decimal or an integer;

    the preparation method comprises the following steps:

    step (a): reacting Trastuzumab with TCEP at a reaction temperature of about 4 ℃ to about 30 ℃ and a pH of about 5.0 to about 6.0;

    step (b): reacting the product of step (a) with a compound of the formula;
12. a method of preparing an antibody drug conjugate, wherein the antibody drug conjugate has a structure represented by the following formula:

    wherein n is 4 to 8, and n is a decimal or an integer;

    the preparation method comprises the following steps:

    step (a): reacting Trastuzumab with TCEP at a reaction temperature of about 25 ℃ and a pH of about 5.6 in a histidine-hydrochloric acid buffer containing EDTA;

    step (b): reacting the product of step (a) with a compound of the formula;

    a step (c): comprising subjecting the product of step (b) to cation chromatography column purification.
13. An antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody drug conjugate is shown as a general formula (Pc-L) _a -Y-D) represents:

    Wherein:

    w is selected from C _1-8 Alkyl radical, C _1-8 alkyl-C _3-7 Cycloalkyl or a linear heteroalkyl of 1 to 8 atoms, said linear heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C is _1-8 Alkyl radical, C _3-7 Cycloalkyl and linear heteroalkyl are each independentlyIs further selected from halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Cycloalkyl substituted with one or more substituents;

    L ² is selected from-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-、-NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-、-S(CH ₂ )p ¹ C (O) -and a chemical bond, wherein p ¹ Is an integer from 1 to 20;

    L ³ is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residue is selected from the group consisting of amino acids of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and optionally further selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 Alkyl, chloro C _1-6 Alkyl, deuterated C _1-6 Alkyl radical, C _1-6 Alkoxy and C _3-7 Substituted with one or more substituents in the cycloalkyl;

    R ¹ is halo C _1-6 Alkyl or C _3-7 A cycloalkyl group;

    R ² selected from hydrogen atoms, halogeno-C _1-6 Alkyl and C _3-7 A cycloalkyl group;

    or, R ¹ And R ² Together with the carbon atom to which they are attached form C _3-7 A cycloalkyl group;

    R ⁵ selected from hydrogen atoms, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

    R ⁶ and R ⁷ Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, deuterated C _1-6 Alkyl and hydroxy C _1-6 An alkyl group;

    m is 0 or 1;

    n is 4 to 8, n is a decimal or an integer;

    pc is an antibody or antigen-binding fragment thereof;

    and, the drug loading distribution of the antibody drug conjugate is: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less;

    preferably, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and in the antibody light chain population, the proportion of antibody light chains which bind 1 drug is 65% or more.
14. An antibody drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate is produced by the production method of the antibody drug conjugate according to any one of claims 1 to 9; and the drug loading distribution of the antibody drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less;

    Preferably, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and the proportion of antibody light chains in the population of antibody light chains that bind 1 drug is 65% or more.
15. An antibody drug conjugate, or a pharmaceutically acceptable salt thereof, wherein the antibody drug conjugate has a structure represented by the formula:

    wherein n is 4 to 8, and n is a decimal or an integer;

    the antibody drug conjugate is prepared by the method for preparing the antibody drug conjugate according to claim 11 or 12; and, the drug loading distribution of the antibody drug conjugate is: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less;

    preferably, the antibody drug conjugate has a drug loading profile of: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind drugs is 5% or less; and the proportion of antibody light chains in the population of antibody light chains that bind 1 drug is 65% or more.