RU2841377C1 - Method of producing an antibody-drug conjugate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: methods for preparing antibody-drug conjugates comprising synthesis and purification steps thereof are disclosed. Anticancer antibody-drug conjugates and pharmaceutically acceptable salts thereof obtained using said methods are also disclosed.

EFFECT: invention provides the improved therapeutic effects of the produced conjugates.

18 cl, 9 dwg, 7 tbl, 72 ex

Description

This application claims priority based on Chinese patent application (application CN 202010219311.2) filed on March 25, 2020 and Chinese patent application (application CN 202110297397.5) filed on March 19, 2021.

AREA OF TECHNOLOGY

The present invention relates to a method for producing an antibody-drug conjugate and, in particular, to the synthesis and purification steps of the method for producing the antibody-drug conjugate.

LEVEL OF TECHNOLOGY

The statements in this section relate only to general information related to the present invention and do not necessarily constitute prior art.

Chemotherapy remains one of the most important antitumor agents, which include surgery, radiation therapy and targeted therapy. Although there are a wide variety of effective cytotoxic drugs, the difference between tumor cells and normal cells is small, which limits the widespread clinical application of these antitumor compounds due to their toxic side effects. Since the antitumor monoclonal antibody has specificity for the surface antigen of tumor cells, antibody-based drugs have become the first-line drugs for antitumor treatment, but when the antibody is used alone as an antitumor drug, the therapeutic effect is often unsatisfactory.

Antibody-drug conjugate (ADC) is formed by linking a monoclonal antibody or antibody fragment to a biologically active cytotoxic drug through a stable chemical linker compound, and can fully utilize the binding specificity of the antibody to the surface antigens of normal cells and tumor cells and the high efficacy of the cytotoxic drug, and avoid the disadvantage of the former component consisting in a weak therapeutic effect, the disadvantage of the latter component consisting in the presence of serious toxic side effects, etc. This means that the antibody-drug conjugate can more precisely bind to tumor cells and have less effect on normal cells compared with conventional chemotherapeutic drugs in the past (Milliard A, (2013) Nature Reviews Drug Discovery, 12:329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceutical Co., Ltd.), the first antibody-drug conjugate, was approved by the US Food and Drug Administration in 2000 for the treatment of acute myelocytic leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

Adcetris® (brentuximab vedotin, Seagen Inc.) was approved under accelerated review by the US FDA in August 2011 for the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778–784; WO2004010957; WO2005001038; US7090843; US7659241; WO2008025020). Adcetris® is a novel ADC that allows the drug to directly target CD30 on lymphoma cells and then undergo endocytosis to induce tumor cell apoptosis.

Both mylotarg and adcetris are targeted therapies for hematological malignancies, which have a relatively simple tissue architecture compared to solid tumors. Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA in February 2013 for the treatment of HER2-positive patients with advanced or metastatic breast cancer who are resistant to trastuzumab (trade name: Herceptin®) and paclitaxel (WO2005037992; US8088387). Kadcyla is the first ADC approved by the US FDA for the treatment of solid tumors.

There are several classes of small molecules with cytotoxicity suitable for antibody drug conjugates, one of which is camptothecin derivatives, which have antitumor activity through topoisomerase I inhibition. The use of a camptothecin derivative, irinotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione), in antibody drug conjugates (ADCs) has been reported as described in WO2014057687; Clinical Cancer Research (2016)22(20):5097–5108; Cancer Sci (2016) 107: 1039–1046. There remains a need for further development of ADC drugs with better therapeutic effects.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the general formula (Pc-L _a -YD):

Where:

W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-C _3-7 cycloalkyl and linear heteroalkyl of 1-8 atoms, wherein the linear heteroalkyl contains 1-3 heteroatoms selected from the group consisting of N, O and S, wherein each of the C _1-8 alkyl, C _3-7 cycloalkyl and linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _1-6 chloroalkyl, deuterated C _1-6 alkyl, C _1-6 alkoxy and C _3-7 cycloalkyl;

L ² is selected from the group consisting of -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, where p ¹ is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _1-6 chloroalkyl, deuterated C _1-6 alkyl, C _1-6 alkoxy and C _3-7 cycloalkyl;

R ¹ is C _1-6 haloalkyl or C _3-7 cycloalkyl;

R ² is selected from the group consisting of hydrogen, C _1-6 haloalkyl and C _3-7 cycloalkyl;

or R ¹ and R ² together with the carbon atoms bound to them form C _3-7 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, deuterated C _1-6 alkyl and hydroxy C _1-6 alkyl;

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, C _1-6 haloalkyl, deuterated C _1-6 alkyl and hydroxy C _1-6 alkyl;

m is 0 or 1;

n is a decimal or integer number between 3 and 8;

RS is an antibody or its antigen-binding fragment;

where the method of obtaining includes the following stages:

step (a): reacting said antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of from about 1°C to about 36°C; and

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

where: W, L ² ,L ³ ,R ¹ ,R ² ,R ⁵ ,R ⁶ ,R ⁷ and m are as defined above.

In another aspect of the present invention, there is provided a method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the following formula:

where n is a decimal or integer number from 4 to 8;

where the method of obtaining includes the following stages:

step (a): reacting said antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of from about 1°C to about 36°C; and

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

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

In an alternative embodiment, the reaction of 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 about 5.0 to about 6.0, and more preferably the reaction is carried out at a pH of about 5.6. In a non-limiting example, 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 a histidine-hydrochloric acid buffer.

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

In an alternative embodiment, the reducing agent in step (a) is selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, 1,4-dimercaptothreitol (DTT), (3-mercaptoethanol ((3-ME)) and other suitable reducing agents, preferably TCEP or a salt thereof, and more preferably tris(2-carboxyethyl)phosphine hydrochloride.

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

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

In an alternative embodiment, the above method of preparation further comprises a step (c) comprising purifying the product of step (b). The purification can be carried out by cationic column chromatography or affinity column chromatography, wherein the cationic column chromatography filling material is selected from the group consisting of Capto S Impact and Poros XS, and preferably Capto S Impact. In some embodiments, Capto S Impact is Capto™ S Impact. In some embodiments, Poros XS is Poros™ XS.

In an alternative embodiment, the drug load (n) may be from 3 to 8, from 4 to 8, or from 5 to 7, preferably from 5.3 to 6.1, and more preferably 5.7, cytotoxic drugs that bind to each 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 an alternative embodiment, the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 4% or less; preferably, the proportion of antibody heavy chains that bind to 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 6% or less. In a non-limiting example, the proportion of antibody heavy chains that bind to 4 drugs is 4% or less, 3% or less, 2% or less, or 1% or less; the proportion of antibody heavy chains that do not bind to drugs is 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Or, in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 65% or more. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 70% or more. In some embodiments, the proportion of antibody heavy chains and/or antibody light chains is determined by reverse phase chromatography.

In an alternative embodiment, the production method is suitable for large-scale production. The amount of the trastuzumab antibody in the production method is 100 mg or more, preferably 1 g or more, more preferably 10 g or more, and most preferably 100 g or more.

In an alternative embodiment, the antibody-drug conjugate has a structure represented by the general formula (Pc-Lb-Y-D):

Where:

s ¹ is an integer between 2 and 8;

Rc, R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ , m and n are as defined above; wherein the method of preparation comprises the following steps:

step (a): reacting said antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of from about 1°C to about 36°C; and

step (b): reacting the product of step (a) with a compound of the following formula (L _b -YD):

where s ¹ , R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and m are as defined above. In an alternative embodiment, the antibody-drug conjugate described above has the following structure:

where Pc and n are as defined in the general formula (Pc-L _a -YD).

In an alternative embodiment, Pc is an antibody or an antigen-binding fragment thereof, and 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 an alternative embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-LeY antigen antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti- SLC44A4, antibodies to mesothelin and their antigen-binding fragment;

Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of trastuzumab, pertuzumab, nimotuzumab, enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, glematumamab and an antigen-binding fragment thereof.

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

where n is a decimal or integer number from 4 to 8.

In another aspect of the present invention, there is provided a method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the following formula:

where n is a decimal or integer number from 4 to 8; where the method of obtaining comprises the following steps:

step (a): reacting trastuzumab with TCEP at a reaction temperature of from about 4°C to about 30°C and a pH of from about 4.5 to about 6.5; and

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

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

where n is a decimal or integer number from 4 to 8; where the method of obtaining comprises the following steps:

step (a): reacting trastuzumab with TCEP at a reaction temperature of approximately 25°C and a pH of approximately 5.6, wherein the reaction is carried out in an EDTA-containing buffer based on histidine and hydrochloric acid;

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

And

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

The present invention also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by the general formula (Pc-L _a -YD):

Where:

W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-C _3-7 cycloalkyl and linear heteroalkyl of 1-8 atoms, wherein the linear heteroalkyl contains 1-3 heteroatoms selected from the group consisting of N, O and S, wherein each of the C _1-8 alkyl, C _3-7 cycloalkyl and linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _1-6 chloroalkyl, deuterated C _1-6 alkyl, C _1-6 alkoxy and C _3-7 cycloalkyl;

L ² is selected from the group consisting of -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, where p ¹ is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _1-6 chloroalkyl, deuterated C _1-6 alkyl, C _1-6 alkoxy and C _3-7 cycloalkyl;

R ¹ is C _1-6 haloalkyl or C _3-7 cycloalkyl;

R ² is selected from the group consisting of hydrogen, C _1-6 haloalkyl and C _3-7 cycloalkyl;

or R ¹ and R ² together with the carbon atoms bound to them form C _3-7 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _1-6 haloalkyl, deuterated C _1-6 alkyl and hydroxy C _1-6 alkyl;

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, C _1-6 haloalkyl, deuterated C _1-6 alkyl and hydroxy C _1-6 alkyl;

m is 0 or 1;

n is a decimal or integer number between 4 and 8;

RS is an antibody or its antigen-binding fragment;

the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less; preferably, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of the antibody heavy chains not binding to drugs is 6% or less. In some embodiments, the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of the antibody heavy chains not binding to drugs is 5% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 65% or more. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 70% or more.

The present invention also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is obtained by the method for producing an antibody-drug conjugate described above; and the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to 4 drugs is 4% or less; preferably, the proportion of antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 6% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 5% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 65% or more. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 70% or more.

The present invention 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:

where n is a decimal or integer number from 4 to 8;

the antibody-drug conjugate is obtained by the method for producing an antibody-drug conjugate described above; and the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less; preferably, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of the antibody heavy chains not binding to drugs is 6% or less. In some embodiments, the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of the antibody heavy chains binding to 4 drugs is 4% or less, and the proportion of the antibody heavy chains not binding to drugs is 5% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 65% or more. In some embodiments, the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to drugs is 4% or less; and in a population of antibody light chains, the proportion of antibody light chains that bind to one drug is 70% or more.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a production method more suitable for large-scale production. In particular, the product obtained by said production method has the advantages of a narrower distribution of drug load, a lower content of free toxin, and a higher yield.

Terms

To facilitate understanding of the present invention, certain technical and scientific terms are separately defined below. Unless otherwise explicitly defined herein, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

Application PCT/CN2019/107873 is incorporated herein by reference in its entirety.

Antibody-drug conjugate (ADC) is formed by linking an antibody or antibody fragment to a biologically active cytotoxin or a small molecule drug with cell killing activity through a stable chemical linker compound, and can fully utilize the specificity of the antibody to tumor cells or the binding specificity of cells with high antigen expression and the high efficiency of the cytotoxin, and avoid the toxic side effect on normal cells. The antibody-drug conjugate can more accurately bind to tumor cells and have less effect on normal cells compared with conventional chemotherapeutic drugs in the past.

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

"Histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloric acid buffer, histidine acetic acid buffer, histidine phosphoric acid buffer, histidine sulfuric acid buffer, etc., and preferably histidine hydrochloric acid buffer, wherein the histidine acetic acid buffer is obtained from histidine and acetic acid, and the histidine hydrochloric acid buffer is obtained from histidine and hydrochloric acid or histidine and histidine hydrochloride.

"Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include sodium hydrogen phosphate and sodium dihydrogen phosphate buffer, sodium hydrogen phosphate and potassium dihydrogen phosphate buffer, sodium hydrogen phosphate and citric acid buffer, etc. A preferred phosphate buffer is sodium hydrogen phosphate and sodium dihydrogen phosphate buffer.

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

The terms "about" and "approximately" as used herein mean that a numerical value is within an acceptable range of error for a particular value as determined by one skilled in the art, and the numerical value depends in part on how the value is measured or determined (i.e., the limits of a measurement system). For example, "about" may mean within 1 or more than 1 standard deviation in accordance with practice accepted in the art. Or "about" or "substantially comprising" may mean a range of up to 20%. Additionally, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the numerical value. When a particular value is specified in this application and the claims, unless otherwise indicated, the meaning of "about" or "substantially comprising" should be presumed to be within the acceptable range of error for that particular value.

The three-letter codes and single-letter codes for amino acids used in this description correspond to those described in J. Biol. Chem, 243, p3558 (1968).

"An antibody" as described herein refers to an immunoglobulin and has a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains of an intact antibody by interchain disulfide bonds. The constant regions of the immunoglobulin heavy chain differ in their amino acid composition and arrangement and, therefore, in their antigenicity. Thus, immunoglobulins can be divided into five classes, which are also called immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same Ig class can be divided into different subclasses according to the difference in the amino acid composition of the hinge regions and the number and position of the disulfide bonds of the heavy chains; For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains according to differences in the constant regions. Each of the five Ig classes can have a κ chain or a λ chain. The antibody described in the present invention is preferably specific antibodies to cell surface antigens on target cells, non-limiting examples of which include one or more of the following: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-LeY antigen antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody, and an anti-mesothelin antibody; the antibody is preferably trastuzumab (trade name: Herceptin), pertuzumab (also known as 2C4, trade name: Perjeta), nimotuzumab (trade name: Taixinsheng®), enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, or glematumamab.

In the heavy and light chains of an antibody, the sequences of approximately 110 amino acids near the N-terminus vary considerably and are therefore called variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are therefore called constant regions. The variable region includes 3 hypervariable regions (HVRs) and 4 framework regions (FRs) with relatively conserved sequences. The 3 hypervariable regions determine the specificity of the antibody and are therefore also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) or heavy chain variable region (HCVR) consists of 3 CDRs and 4 FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The 3 CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; and the 3 CDRs of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. The amino acid residues of the CDR regions of the LCVR and HCVR regions of the antibodies or antigen-binding fragments described herein correspond to the known numbering scheme according to Kabat (LCDR 13, HCDR 13) with respect to number and positions.

In the present invention, the light chain of the antibody of the present invention may further comprise a light chain constant region comprising human or murine κ and λ chains or variants thereof.

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

The antibody of the present invention includes a murine antibody, a chimeric antibody and a humanized antibody, and preferably a humanized antibody.

The term "mouse antibody" as used herein refers to an antibody obtained from mice in accordance with the knowledge and skills in the art. In the process of obtaining, a test subject is administered a certain antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.

The term "chimeric antibody" refers to an antibody obtained by fusing the variable region of a mouse antibody and the constant region of a human antibody, which can reduce the immune response caused by the mouse antibody. To obtain a chimeric antibody, first a hybridoma secreting a mouse-specific monoclonal antibody is created, then the variable region gene is cloned from the mouse hybridoma cells, and then the constant region gene of a human antibody is cloned, if necessary, the mouse variable region gene is linked to the human constant region gene to form a chimeric gene, the chimeric gene is inserted into an expression vector, and finally the chimeric antibody molecules are expressed in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences onto the variable region framework of a human antibody, i.e., a different type of framework sequence, human. Such an antibody can overcome the heterogeneous response caused by a chimeric antibody due to the presence of a large number of murine protein components. Such framework sequences can be obtained from publicly available DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of the human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the FR sequences in the variable region of the human antibody can be subjected to minimal reversible mutations or back mutations to maintain activity. The humanized antibody of the present invention also includes humanized antibodies further subjected to CDR affinity maturation 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.

The "antigen-binding fragment of an antibody" described herein may refer to Fab fragments, Fab' fragments, and F(ab') ₂ fragments that have antigen-binding activity, and Fv fragments and scFv fragments that bind to an antigen. An Fv fragment comprises the variable region of the heavy chain and the variable region of the light chain of an antibody, but not the constant region, and is the smallest fragment of an antibody with all antigen-binding sites. Typically, an Fv antibody also comprises a polypeptide linker between the VH and VL domains and is capable of forming the structure necessary for antigen binding. Two variable regions of an antibody may also be linked into a single polypeptide chain using various linkers, known as a single-chain antibody or single-chain fv (sFv).

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

"ADCC", i.e. antibody-dependent cell-mediated cytotoxicity, as described herein, refers to the direct killing of antibody-coated target cells by cells expressing an Fc receptor through recognition of the Fc segment of the antibody. The ADCC effector function of an antibody can be reduced or eliminated by modification of the Fc segment of IgG. The modification refers to a mutation in the constant region of the heavy chain of the antibody, such as a mutation selected from the group consisting of N297A, L234A and L235A of IgG1; IgG2/4 chimeras; and F234A/L235A of IgG4.

"Mutations" in the mutant sequences described herein include, but are not limited to, "backmutation," "conservative modification," or "conservative substitution or replacement." A "conservative modification" or "conservative substitution or replacement" as described herein refers to the replacement of amino acids in a protein with other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, or main chain conformation and rigidity) so that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art will recognize that, in general, substitution of a single amino acid in non-essential regions of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p224, (4th edition)). Furthermore, substitution with amino acids of similar structure or function is unlikely to affect biological activity.

The term "mutant sequence" as described herein refers to a nucleotide sequence and/or an amino acid sequence having a different degree of percent identity to the nucleotide sequence and/or amino acid sequence of the present invention when mutational modifications such as substitutions, insertions or deletions are suitably introduced into the nucleotide sequence and/or amino acid sequence of the present invention. The sequence identity described herein may be at least 85%, 90% or 95%, and preferably at least 95%. Non-limiting examples of sequence identity include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. Sequence comparison and determination of the percent identity of two sequences can be accomplished using the default settings for the BLASTN/BLASTP algorithm available from the National Center for Biotechnology Information website.

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

The linker includes extender units, spacer units and amino acid units and can be synthesized by methods known in the art, such as those described in US2005-0238649 A1. The linker can be a "cleavable linker" that facilitates the release of drugs in cells. 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 Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

The engineered antibody or antigen-binding fragment thereof of the present invention can be produced and purified by conventional techniques. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems can glycosylate antibodies, particularly at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free antibody bioreactor medium. The secreted antibody culture can be purified by conventional techniques, for example, purification is performed on an A- or G-Sepharose FF column containing an adjusted buffer. Non-specifically bound fractions are washed away. The bound antibody is then eluted by a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

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

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

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having 2 residues derived from a parent alkane by removing two hydrogen atoms from the same or two different carbon atoms. It is a linear or branched group containing from 1 to 20 carbon atoms, preferably alkylene containing from 1 to 12 carbon atoms, and more preferably alkylene containing from 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene( _-CH2- ), 1,1-ethylidene(-CH( _{CH3 )} -), 1,2-ethylidene( _-CH2CH2 )-, 1,1-propylidene(-CH( _CH2CH3 )-), _1,2 -propylidene( _-CH2CH ( _CH3 )-), 1,3-propylidene( _{-CH2CH2CH2- )} , _1,4 - _butylidene ( _{-CH2CH2CH2CH2- )} , _{1,5 - butylidene(-CH2CH2CH2CH2CH2- )} , etc. The alkylene may _be substituted or unsubstituted _. When substituting, the substituent may be substituted at any available site on the compound with one or more substituents, preferably independently optionally selected from the group consisting of alkyl, 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 include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy and cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. The cycloalkyl ring contains 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 include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyl includes spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing from 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen and S(O) _m (where m is an integer from 0 to 2), excluding the cyclic portion -O-O-, -OS- or -SS-, and the remaining ring atoms are carbon atoms. Heterocycloalkyl preferably contains from 3 to 12 ring atoms, of which from 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains from 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Non-limiting examples of polycyclic heterocyclyl include spiroheterocyclyl, fused heterocyclyl, and bridged heterocyclyl.

The term "spiroheterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl group in which the monocyclic rings have one atom in common (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. These rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system. Preferably, the bridged heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. Depending on the number of spiro atoms common to the rings, spiroheterocyclyl may be monospiroheterocyclyl, bispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and bispiroheterocyclyl, more preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl. Non-limiting examples of spiroheterocyclyl include:

The term "fused heterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring shares a pair of adjacent atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Preferably, the bridged heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. Depending on the number of rings formed, the fused heterocyclyl may be a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, preferably a bicyclic or tricyclic fused heterocyclyl, and more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclyl. Non-limiting examples of fused heterocyclyl include:

The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings have two common atoms that are not directly bonded to each other, wherein these rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Preferably, the bridged heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. Depending on the number of rings formed, the bridged heterocyclyl can be a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, preferably a bicyclic, tricyclic or tetracyclic bridged heterocyclyl and more preferably a bicyclic or tricyclic bridged heterocyclyl. Non-limiting examples of bridged heterocyclyl include:

A heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring bound to the parent structure is heterocyclyl. Non-limiting examples of a heterocyclyl ring include, but are not limited to:

etc.

Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of 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, preferably 6- to 10-membered carbon monocyclic or fused polycyclic (i.e., where the rings share a pair of adjacent carbon atoms) group having a conjugated π-electron system, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, where the ring bound to the parent structure is an aryl ring. Non-limiting examples of an aryl ring include, but are not limited to:

Aryl may be substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of 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 containing from 1 to 4 heteroatoms and from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. Heteroaryl is preferably 5-10 membered, more preferably 5- or 6-membered, for example, furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and tetrazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bound to the parent structure is a heteroaryl ring. Non-limiting examples of a hetero aryl ring include:

Heteroaryl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of 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" refers to a group that can be easily removed and is intended to protect the amino group from being altered when a reaction is carried out elsewhere in the molecule. Non-limiting examples of the amino protecting group include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, etc. These groups may be optionally substituted with 1 to 3 substituents selected from the group consisting of halogen, alkoxy, and nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

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

The term "haloalkyl" refers to alkyl substituted with one or more halogen atoms, wherein alkyl is as defined above.

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

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

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

The term "amino" refers to -NH ₂ .

The term "nitro" refers to -NO ₂ .

The term "cyano" refers to -CN.

The term "optional" or "optionally" means that the event or circumstance described below may, but does not necessarily have to, occur, and that the description includes instances where the event or circumstance does or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of certain sequences may be present, but are not necessarily present.

The term "substituted" means that one or more, preferably up to 5, more preferably from 1 to 3 hydrogen atoms in the group are independently replaced by a corresponding number of substituents. It is obvious that the substituent is located only in its possible chemical positions, and those skilled in the art will be able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, a substitution may be unstable when an amino or hydroxy group having a free hydrogen is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

The term "drug load" (DAR) refers to the average amount of cytotoxic drug loaded per antibody or antigen-binding fragment thereof in an ADC, and can also be represented as a drug-to-antibody ratio, which is an integer or decimal number. In embodiments of the present invention, the drug load is represented as n, and exemplary values can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drug per ADC molecule after binding reactions can be characterized by conventional methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC.

The term "drug load distribution" refers to the distribution of antibodies bound to different numbers of drugs in a population of antibody-drug conjugates, such as the distribution of antibodies bound to 0, 2, 4, 6 and 8 drugs in a population. Wherein DARs comprising 1, 3, 5 and 7 drugs may also be included in the mixture due to the potential formation of degradation products. In the present invention, the drug load distribution of antibodies can be characterized by the heavy chains of the antibody binding to different numbers of drugs, such as: H ₀ is a heavy chain that does not bind drugs, H ₁ is a heavy chain that binds one drug, H ₂ is a heavy chain that binds two drugs, H ₃ is a heavy chain that binds three drugs, and H ₄ is a heavy chain that binds four drugs. For illustration, a _H3 fraction of 4% means that in a population of heavy chains of antibody-drug conjugates, the fraction of heavy chains binding to three drugs is 4%. Accordingly, the drug load distribution of antibodies in the present invention can also be characterized by antibody light chains binding to different numbers of drugs, where _L0 is an antibody light chain that does not bind drugs and _L1 is an antibody light chain that binds to one drug.

"Administering," "administering," and "treating," when used with animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with said animals, humans, subjects, cells, tissues, organs, or biological fluids. "Administering," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treating cells includes contacting a reagent with cells, as well as contacting a reagent with a fluid, where the fluid is in contact with the cells. "Administering," "administering," and "treating" also refers to treating, for example, a cell with a reagent, diagnostic, binding composition, or another cell in vitro and ex vivo. "Treatment" when applied to humans, veterinary subjects, or research subjects refers to therapeutic treatment, preventive or prophylactic measures, and investigative and diagnostic uses.

"Treatment" or "treatment" refers to the administration of a therapeutic agent, such as a composition comprising any of the conjugated compounds of the present invention, internally or extracorporeally to a patient who has one or more symptoms of a disease in which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, to cause a regression of such symptoms or to suppress the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate the symptoms of the disease (also referred to as a "therapeutically effective amount") may vary depending on various factors, such as the pathological condition, the age and body weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a symptom of the disease has been alleviated may be assessed using any of the clinical testing methods commonly used by physicians or other healthcare professionals to assess the severity or progression of the symptom. Although embodiments of the present invention (e.g., treatment methods or products) may not be effective in alleviating the symptoms of every disease of interest, they should alleviate the symptoms of the disease of interest in a statistically significant number of patients, as determined by any statistical testing methods known in the art, such as the Student t-test, the chi-square test, the Mann-Whitney U-test, the Kruskal-Wallis (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

"An effective amount" includes an amount sufficient to alleviate or prevent a symptom or medical condition. An effective amount also refers to an amount sufficient to provide or facilitate a diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route, route and dosage of administration, and the severity of adverse effects. An effective amount may be the maximum dose or schedule of administration that avoids significant adverse effects or toxic effects.

"Exchange" refers to the replacement of a solvent system that solubilizes the antibody protein. For example, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a buffer system of stable composition by physical operations, such that the antibody protein is present in a stable composition. Such physical operations include, but are not limited to, ultrafiltration, dialysis, or centrifugal recovery.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1A shows the results of a plasma stability test for ADC-19 of the present invention.

FIG. 1B shows the results of a plasma stability test for ADC-18 of the present invention.

FIG. 1C shows the results of a plasma stability test for ADC-20 according to the present invention.

FIG. 2 shows the efficacy evaluation of ADC-21 and ADC-24 in LMT-1 tumor-bearing mice.

FIG. 3 shows the evaluation of the therapeutic effect of ADC in nude mice bearing SK-BR-3 human breast cancer cell xenograft.

FIG. 4 shows the results of a plasma stability test for ADC-25 according to the present invention.

FIG. 5 shows the therapeutic effect of ADC in nude mice bearing human brain astrocytoma U87MG xenograft.

FIG. 6 shows the therapeutic effect of ADC in nude mice bearing a Detroit 562 cell xenograft of human pharyngeal cancer metastasizing to pleural effusion.

FIG. 7 shows the therapeutic effect of ADC in nude mice bearing human U87MG glioblastoma xenograft.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described below with reference to examples, but these examples do not limit the scope of the present invention.

Experimental procedures for which no specific conditions are indicated in the examples of the present invention are generally carried out under conventional conditions or under conditions recommended by the manufacturer of the starting materials or commercial products. Reagents not specifically indicated are commercially available conventional reagents.

I. Preparation of antibody-drug conjugate and its properties and biological testing The structures of the compounds were determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument using deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ) and deuterated methanol ( _CD3OD ) as detection solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in units of ^10-6 (ppm).

MS spectra were measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

UHPLC analysis was performed using a Waters Acquity UPLC SQD liquid chromatography/tandem mass spectrometry system.

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

UV-HPLC analysis was performed using a Thermo nanodrop2000 UV spectrophotometer.

The proliferation inhibition rates and IC ₅₀ values were measured using a PHERA starFS microplate reader (BMG, Germany).

Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates with sizes from 0.15 mm to 0.2 mm were selected for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

Yantai Huanghai silica gel of 200-300 mesh was generally used as a carrier for column chromatography.

The known starting materials described herein can be synthesized by methods known in the art or can be purchased from ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, Chembee Chemicals, etc.

In the Examples, reactions were carried out under argon atmosphere or nitrogen atmosphere unless otherwise stated.

An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to a cylinder containing approximately 1 L of argon or nitrogen.

Hydrogen atmosphere means that the reaction flask is connected to a cylinder containing approximately 1 L of hydrogen.

A Parr 3916EKX hydrogenator, a Qinglan QL-500 hydrogenator, or an HC2-SS hydrogenator were used in the pressure hydrogenation reactions.

Hydrogenation reactions typically involve 3 cycles of vacuum and hydrogen purge.

A microwave reactor CEM Discover-S 908860 was used for microwave-assisted reactions.

In the Examples, the solution in the reaction refers to an aqueous solution unless otherwise stated.

In the Examples, the reaction temperature is room temperature unless otherwise stated.

Room temperature is the optimum reaction temperature, which ranges from 20°C to 30°C.

Preparation of PBS buffer with pH 6.5 in examples: 8.5 g _KH2PO4 , 8.56 g _{K2HPO4⋅3H2O} , 5.85 g NaCl and 1.5 g EDTA _were added to _a flask and the _volume was adjusted to 2 L. All additives were dissolved by ultrasound and the solution was mixed well by shaking to obtain the desired buffer.

The eluent system for column chromatography and the developing solvent system for thin layer chromatography used for the purification of the compound were as follows: A: dichloromethane-isopropanol system, B: dichloromethane-methanol system, and C: petroleum ether-ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound or by adding a small amount of triethylamine and an acidic or basic reagent.

Some of the compounds of the present invention were characterized by Q-TOF LC-MS (tandem quadrupole-time-of-flight liquid chromatography-mass spectrometry). An Agilent 6530 true mass quadrupole-time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1 x 75 mm chromatography column) were used in the Q-TOF LC-MS analysis.

Example 1-1

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

To exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686 A1") was added 1 ml of N,N-dimethylformamide. The mixture was cooled to 0-5°C in an ice water bath, followed by the addition of a drop of triethylamine. The reaction solution was stirred until it became clear. To the reaction solution were successively added 1-hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol). After completion of the addition, the reaction solution was stirred at 0-5°C for 2 h, quenched with 5 ml of water and extracted with ethyl acetate (8 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, yield 82.1%).

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

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.90-7.84 (m, 1H), 7.80-7.68 (m, 1H), 5.80-5.70 (m, 1H), 5.62-5.54 (m, 2H), 5.44-5.32 (m, 2H), 5.28-5.10 (m, 2H), 3.40-3.15 (m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1.75 (m, 2H), 1.68-1.56 (m, 1H), 1.22-1.18 (m, 2H), 1.04-0.98 (m, 2H), 0.89 (t, 3H).

Example 1-2

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

To 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The mixture was purged with argon three times and cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction solution was stirred until it became transparent. To the reaction solution were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, obtained according to the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After completion of the addition, the reaction solution was stirred at 0-5°C for 1 h. The ice water bath was removed, the reaction solution was heated to 30°C, stirred for 2 h, and concentrated under reduced pressure. The obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (2-A: 1.5 mg, 2-B: 1.5 mg).

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

Compound in the single 2-B configuration (shorter retention time) UHPLC analysis: retention time: 1.06 min; purity: 88% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 x 50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2.14 (m, 1H), 1.98 (q, 2H), 1.87-1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H).

Compound in the single configuration 2-A (longer retention time) UHPLC analysis: retention time: 1.10 min; purity: 86% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

Example 1-3

(S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-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 (5.0 mg, 9.41 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction solution was stirred until it became transparent. To the reaction solution were added 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially. After completion of the addition, the reaction solution was stirred at 0-5 °C for 10 min. The ice water bath was removed, the reaction solution was heated to 30 °C, stirred for 8 h, and concentrated under reduced pressure. The obtained crude compound 3 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).

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

Compound in a single configuration (shorter retention time) UHPLC analysis: retention time: 1.11 min; purity: 88% (chromatographic column: ACQUITY UPLC VENC18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.26-2.20 (m, 1H), 2.16-2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Compound in a single configuration (longer retention time) UHPLC analysis: retention time: 1.19 min; purity: 90% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.22-2.14 (m, 1H), 2.04-1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Example 1-4

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

To 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine. The reaction solution was stirred until it became transparent. 1-Hydroxycyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, obtained according to the method disclosed in patent application “WO2013106717”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added to the reaction solution successively. After the completion of the addition, the reaction solution was stirred at 0-5 °C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 4 (2.5 mg, yield 80.9%).

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

^1H NMR (400 MHz, CDCl ₃ ): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H), 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2.08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

Example 1-5

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

To 1b (2.0 mg, 3.76 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine. The reaction solution was stirred until it became transparent. 1-Hydroxycyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, obtained according to the method disclosed in patent application “WO201396771”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were added to the reaction solution successively. After the completion of the addition, the reaction solution was stirred at 0-5 °C for 2 h, quenched with 5 mL of water and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 5 (1.0 mg, yield 50%).

MC m/z (ESI): 533?9.[M+1].

^1H NMR (400 MHz, CDCl ₃ ): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55-6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3.30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35-2.28 (m, 1H), 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

Example 1-6

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

To 1b (3.0 mg, 5.64 μmol) was added 1 ml of N,N-dimethylformamide. The mixture was cooled to 0-5°C in an ice water bath followed by the addition of a drop of triethylamine.

The reaction solution was stirred until it became transparent. To the reaction solution were added sequentially 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, obtained according to the method described in the document “Journal of the American Chemical Society, 2014, vol. 136, no. 22, p. 8138-8142”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After completion of the addition, the reaction solution was stirred at 0-5 °C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 6 (2.1 mg, yield 67.9%).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 7.85-7.62 (m, 1H), 6.88 (br, 1H), 5.87-5.48 (m, 2H), 5.47-5.33 (m, 1H), 5.31-5.06 (m, 1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

Example 1-7

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

To 1b (3.0 mg, 5.64 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction solution was stirred until it became transparent. To the reaction solution were added 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplier PharmaBlock), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) sequentially. After completion of the addition, the reaction solution was stirred at 0-5 °C for 10 min. The ice water bath was removed, the reaction was stirred at room temperature for 2 h and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 7 (2.5 mg, yield 83.1%).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3.17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 (m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0.83 (m, 4H).

Example 1-8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-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

Stage 1

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

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

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

Stage 2

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

8c (50 mg, 0.10 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (v:v=2:1), and a carbon-supported palladium catalyst (25 mg, 10% loading) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 h. The reaction solution was filtered through celite, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 8d (41 mg, 100% yield).

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

Stage 3

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

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

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

Stage 4

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

8e (4 mg, 4.84 μmol) was dissolved in 0.2 ml of dichloromethane and 0.1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 8f (2.9 mg), which was directly used in the next step without purification.

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

Stage 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-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 (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times and cooled to 0-5 °C in an ice water bath, followed by the addition of a solution of (S)-2-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 mmol, prepared according to the method disclosed in patent application "EP2907824)) in 0.3 mL of N,N-dimethylformamide, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction solution was stirred in an ice bath for 30 min, followed by an ice bath. The reaction solution was warmed to room temperature and stirred for 15 min, and purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAC), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield 39.0%).

MCm/z (ESI): 1060.0.[M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m, 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4.52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47(m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H), 2.12-1.93 (m, 4H), 1.90-1.74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

Example 1-9

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6.7 ]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-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

Stage 1

Benzyl 2-cyclopropyl 2-hydroxyacetate 9a 2a (1.3 g, 11.2 mmol; prepared according to the method disclosed in patent application "WO2013/106717") was dissolved in 50 mL of acetonitrile and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added successively. The reaction solution was stirred at room temperature for 48 h, filtered through celite and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 9a (2 mg, yield 86.9%).

Stage 2

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

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

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

Stage 3

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

9b (20 mg, 0.038 mmol) was dissolved in 4.5 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (v:v=2:1), and palladium catalyst on carbon support (12 mg, 10% loading, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 h. The reaction solution was filtered through celite, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to give the crude title product 9c (13 mg), which was directly used in the next step without purification.

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

Stage 4

(9H-fluoren-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa hydro-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

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

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

Stage 5

2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-diox o-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 (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 h and concentrated under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left. Then, the supernatant was removed and the solid was saved. The solid residue was concentrated under reduced pressure and dried with an oil pump to give the crude title product 9e (17 mg), which was directly used in the next step without purification.

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

Stage 6

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6.7 ]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-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 (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and a solution of 8g (21.2 mg, 44.8 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The reaction solution was stirred in an ice bath for 10 min, after which the ice bath was The reaction solution was warmed to room temperature and stirred for 1 h to obtain compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).

MCm/z (ESI): 1074.4.[M+1].

Compound in the single configuration 9-A (shorter retention time) UHPLC analysis: retention time: 1.14 min; purity: 85% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 1H), 2.80-2.62 (m, 1H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H). Compound in the single configuration 9-B (longer retention time) UHPLC analysis: retention time: 1.16 min; purity: 89% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 3H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

Example 1-10

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

Stage 1

Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

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

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

Stage 2

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

8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to the reaction flask, followed by the addition of 3 mL of tetrahydrofuran. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and potassium tert-butoxide (38 mg, 0.34 mmol) was added. The ice bath was removed, the reaction solution was warmed to room temperature and stirred for 20 min, 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated, the resulting residue was dissolved in 2 ml of dioxane and 0.4 ml of water was added, followed by the addition of sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol). The reaction solution was stirred at room temperature for 1 h, 20 ml of water was added and extracted with ethyl acetate (10 ml × 3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using a developing solvent system C to give the title product 10b (51 mg, yield 55.3%).

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

Stage 3

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

10b (15 mg, 0.28 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (v:v=2:1), and a carbon-supported palladium catalyst (15 mg, 10% loading) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 h. The reaction solution was filtered through celite, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 10c (13 mg).

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

Stage 4

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

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

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

Stage 5

2-((2-Aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di oxo-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 (16 mg, 18.4 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left. Then, the supernatant was removed and the solid was saved; these procedures were repeated three times. The solid residue was concentrated under reduced pressure and dried with an oil pump to give the crude title product 10e (12 mg), which was directly used in the next step without purification.

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

Stage 6

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

Crude 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and a solution of 8g (14 mg, 29.6 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 mmol). The reaction solution was stirred in an ice bath for 30 min, after which the ice bath was The reaction solution was warmed to room temperature and stirred for 1 h to obtain compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to obtain the title product (2.7 mg, 2.6 mg).

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

Compound in a single configuration (shorter retention time) UHPLC analysis: retention time: 1.18 min; purity: 91% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.97 (d, 1H), 8.85-8.76 (m, 1H), 8.37-8.27 (m, 1H), 8.12-8.02 (m, 1H), 8.02-7.95 (m, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.26-7.10 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.52 (s, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 1H), 5.37-5.25 (m, 3H), 5.23-5.13 (m, 1H), 4.81-4.68 (m, 2H), 4.51-4.41 (m, 1H), 3.78-3.45 (m, 6H), 3.21-3.13 (m, 1H), 3.02-2.93 (m, 1H), 2.77-2.63 (m, 2H), 2.45-2.29 (m, 3H), 2.24-2.05 (m, 3H), 2.04-1.93 (m, 5H), 1.90-1.75 (m, 2H), 1.52-1.38 (m, 4H), 0.90-0.78 (m, 5H).

Compound in a single configuration (longer retention time) UHPLC analysis: retention time: 1.23 min; purity: 90% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.05 (d, 1H), 8.97-8.88 (m, 1H), 8.35-8.27 (m, 1H), 8.11-8.03 (m, 1H), 8.02-7.95 (m, 1H), 7.80 (d, 1H), 7.34 (s, 1H), 7.29-7.13 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.54 (s, 1H), 5.64-5.55 (m, 1H), 5.43 (s, 1H), 5.36-5.20 (m, 3H), 4.92-4.85 (m, 1H), 4.82-4.72 (m, 2H), 4.52-4.42 (m, 1H), 3.77-3.48 (m, 6H), 3.21-3.14 (m, 1H), 3.03-2.95 (m, 1H), 2.79-2.65 (m, 2H), 2.47-2.28 (m, 3H), 2.25-2.05 (m, 3H), 2.05-1.94 (m, 5H), 1.91-1.76 (m, 2H), 1.52-1.37 (m, 4H), 0.92-0.77 (m, 5H).

Example 1-11

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-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

Stage 1

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

Benzyl 1-hydroxycyclobutanecarboxylate 11a (167 mg, 0.81 mmol, prepared according to the method described in the document "Journal of Medicinal Chemistry, 2013, vol. 56, no. 13, pp. 5541-5552") and 8b (150 mg, 0.41 mmol) were added to the reaction flask, followed by the addition of 5 mL of tetrahydrofuran. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and potassium tert-butoxide (92 mg, 0.82 mmol) was added. The ice bath was removed, the reaction solution was warmed to room temperature and stirred for 10 min, 20 ml of ice water was added and extracted with ethyl acetate (5 ml × 2) and chloroform (5 ml × 5). The organic phases were combined and concentrated, the resulting residue was dissolved in 3 ml of dioxane and 0.6 ml of water was added, followed by the addition of sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol). The reaction solution was stirred at room temperature for 1 h, 20 ml of water was added and extracted with ethyl acetate (8 ml × 3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 11b (37 mg, yield 17.6%).

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

Stage 2

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

11b (37 mg, 71.9 μmol) was dissolved in 3 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (v:v=2:1), and a carbon-supported palladium catalyst (15 mg, 10% loading) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 2 h. The reaction solution was filtered through celite, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 11c (35 mg, 82% yield), which was directly used in the next step.

Stage 3

(9H-fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclobutoxy)methyl)amino)-2-oxoethyl)carbamate 11d 1b (10 mg, 0.018 mmol) was added to the reaction flask, followed by the addition of 1 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times and cooled to 0-5 °C in an ice-water bath, followed by the addition of a drop of triethylamine, a solution of 11 c (13 mg, 0.031 mmol) in 0.5 ml N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol). The reaction solution was stirred in an ice-water bath for 40 min, 8 ml of water was added and extracted with ethyl acetate (5 ml × 3). The organic phase was washed with saturated sodium chloride solution (8 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography with developing solvent system A to give the title product 11d (19 mg, yield 73.9%).

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

Stage 4

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

11d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 4 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 11e (15 mg), which was directly used in the next step without purification.

Stage 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-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 (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and a solution of 8 g (1.5 mg, 3.17 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction solution was stirred at room temperature for 30 min and concentrated to dryness using an oil pump to remove DMF, and the residue was dissolved in DCM and purified twice directly by thin layer chromatography (developing solvent polarity: DCM/MeOH = 10/1) to give the title product 11 (1 mg, yield 28.8%).

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

^1H NMR (400 MHz, CDCl ₃ ): δ 8.70-8.60 (m, 1H), 8.28-8.19 (m, 1H), 8.13-7.91 (m, 3H), 7.79-7.71 (d, 1H), 7.29 (s, 1H), 7.25-7.09 (m, 4H), 6.98 (s, 1H), 6.71-6.62 (m, 1H), 6.55-6.47 (m, 1H), 5.64-5.54 (m, 2H), 5.40 (s, 1H), 5.35-5.27 (t, 2H), 5.17-5.10 (m, 2H), 4.60-4.51 (m, 1H), 4.51-4.35 (m, 2H), 3.93-3.78 (m, 3H), 3.71-3.59 (m, 3H), 3.01-2.88 (m, 3H), 2.70-2.64 (m, 2H), 2.44-2.30 (m, 3H), 2.28-2.14 (m, 3H), 2.11-1.92 (m, 6H), 1.90-1.76 (m, 3H), 1.51-1.39 (m, 4H), 0.92-0.75 (m, 6H).

Example 1-12

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

Stage 1

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

Stage 2

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

To 1b (45 mg, 0.085 mmol) were added 1.5 ml of ethanol and 1.5 ml of N,N-dimethylformamide, the reaction solution was purged with argon three times, 0.1 ml of N-methylmorpholine was added dropwise and stirred until the solution became transparent. Compound 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were added to the reaction solution successively. After completion of the addition, the reaction solution was stirred at room temperature for 3 h and concentrated under reduced pressure. The obtained crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatographic column: Sharpsil-T C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAC), B-acetonitrile, gradient elution, flow rate: 18 ml/min) to give the title product (7 mg, 15 mg).

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

Compound in a single configuration (shorter retention time) UHPLC analysis: retention time: 1.345 min; purity: 72% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1 x 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.42 (d, 1H), 7.78 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.60-5.50 (m, 2H), 5.42 (s, 1H), 5.19 (q, 2H), 4.02-4.00 (m, 1H), 3.21-3.11 (m, 2H), 2.39 (s, 3H), 2.21-2.07 (m, 2H), 2.05-1.95 (m, 1H), 1.92-1.68 (m, 4H), 1.53-1.41 (m, 1H), 0.87 (t, 3H), 0.48-0.34 (m, 2H), 0.14-0.01 (m, 2H).

Compound in a single configuration (longer retention time) UHPLC analysis: retention time: 1.399 min; purity: 88% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1 x 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.36 (d, 1H), 7.77 (d, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.58-5.51 (m, 1H), 5.48 (d, 1H), 5.42 (s, 1H),5.20 (q, 2H), 4.09-4.02 (m, 1H), 3.22-3.11 (m, 2H), 2.39 (s, 3H), 2.27-2.06 (m, 2H), 2.05-1.95 (m, 1H), 1.93-1.81 (m, 2H), 1.65-1.43 (m, 2H), 1.32-1.21 (m, 1H), 0.87 (t, 3H), 0.48-0.33 (m, 2H), 0.14-0.01 (m, 2H).

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

The title compound 13 was prepared by the method disclosed in Example 76 on page 147 of the patent specification EP2907824A1.

Example 1-14

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

Stage 1

Benzyl 3-cyclopropyl 2-hydroxypropionate 14a

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

Stage 2

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

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

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

Stage 3

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

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

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

Stage 4

(9H-fluoren-9-yl)methyl(2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl l-10,13-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

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

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

Stage 5

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 14th

14d (29 mg, 33.9 μmol) was dissolved in 0.8 ml of dichloromethane, and 0.4 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left alone. Then, the supernatant was removed; and these procedures were repeated three times. The residue was concentrated under reduced pressure and dried with an oil pump to give the crude title product 14e (22 mg), which was directly used in the next step without purification.

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

Stage 6

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

Crude 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and 8 g (24 mg, 50.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (14 mg, 50.6 μmol) were added successively, and the ice bath was removed. The reaction solution was warmed to room temperature and stirred for 1 h to obtain compound 14. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to obtain the title product (2 mg, 2 mg).

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

Compound in a single configuration (shorter retention time) UHPLC analysis: retention time: 1.18 min; purity: 88% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

Compound in a single configuration (longer retention time) UHPLC analysis: retention time: 1.23 min; purity: 96% (chromatographic column: ACQUITY UPLC BENS18 1.7 μm 2.1 × 50 mm; mobile phase: A-water (5 mmol NH ₄ OAC), B-acetonitrile).

Example 1-15

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

Stage 1

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

To the reaction flask were added compound 8b (500 mg, 1.35 mmol), 6 ml of tetrahydrofuran and benzyl 1-hydroxymethylcyclopropane-1-carbamate 15a (233 mg, 1.13 mmol; prepared by the method disclosed in “Example 22-2 on page 262 of the specification of patent application EP2862856 A1”). The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, and sodium hydride (54 mg, 1.35 mmol) was added, and the ice bath was removed. The reaction solution was warmed to room temperature and stirred for 40 min, then cooled to 0 °C, 20 ml of ice water was added and extracted with ethyl acetate (5 ml × 2) and chloroform (5 ml × 5). The organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography with developing solvent system B to give the title product 15b (15 mg, yield 2.5%).

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

Stage 2

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

15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate and palladium catalyst on carbon support (3 mg, 10% loading, dry) was added. The reaction solution was purged with hydrogen three times, stirred at room temperature for 4.5 h, filtered through celite and the filter cake was washed with ethyl acetate. The filtrate was concentrated to give the title product 15c (11 mg, 89% yield).

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

Stage 3

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

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

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

Stage 4

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

15d (19 mg, 22.56 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 h and concentrated under reduced pressure at 0 °C. 1 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 15e (13.9 mg), which was directly used in the next step without purification.

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

Stage 5

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-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 (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide. The reaction solution was purged with argon three times, cooled to 0-5°C in an ice water bath, and 8 g (15.8 mg, 33.4 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol) were added. The reaction solution was warmed to room temperature and stirred for 60 min, and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAC), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 15 (2.5 mg, 10.3% yield).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.51-8.37 (m, 1H), 8.22 (t, 1H), 8.14-8.02 (m, 2H), 8.011-7.94 (m, 1H), 7.82-7.73 (m, 1H), 7.29 (s, 1H), 7.26-7.10 (m, 3H), 6.98 (s, 1H), 6.53-6.47 (m, 1H), 5.62-5.50 (m, 1H), 5.45-5.36 (m, 1H), 5.35-5.23 (m, 2H), 5.13-5.02 (m, 2H), 4.61-4.50 (m, 2H), 4.42-4.28 (m, 2H), 3.76-3.61 (m, 3H), 3.60-3.45 (m, 3H), 3.27-3.23 (m, 1H), 3.20-2.81 (m, 7H), 2.75-2.61 (m, 3H), 241-2.28 (m, 3H), 2.23-2.13 (m, 2H), 2.11-2.01 (m, 1H), 2.03-1.94 (m, 1H), 1.90 (s, 1H), 1.87-1.74 (m, 2H), 1.53-1.36 (m, 3H), 1.29-1.08 (m, 4H), 0.90-0.68 (m, 4H).

Example 1-16

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

Stage 1

1-(hydroxymethyl)cyclobutane-1-carboxylic acid 16b Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier Alfa) was dissolved in methanol (2 mL) and water (1 mL), and sodium hydroxide (126 mg, 3.15 mmol) was added. The reaction solution was heated to 40 °C, stirred for 3 h, cooled to room temperature, and concentrated under reduced pressure to remove the organic solvent. The reaction solution was back-extracted with diethyl ether (10 mL) to collect the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 6 N aqueous hydrochloric acid and concentrated under reduced pressure to give a solid. Toluene (3 mL) was added, followed by concentration under reduced pressure to dryness; the procedures were repeated three times.

The residue was dried using an oil pump to give the crude title product 16b (206 mg), which was used directly in the next step without purification.

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

Stage 2

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

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

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

Stage 3

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

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

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

Stage 4

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

16d (12 mg, 22.7 μmol) was dissolved in 1×5 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (v:v=2:1), and a carbon-supported palladium catalyst (5 mg, 10% loading) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 2 h. The reaction solution was filtered through celite, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give the crude title product 16e (10 mg), which was directly used in the next step without purification.

Stage 5

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

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

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

Stage 6

1-(((2-Aminoacetylamino)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16g, 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 ml of dichloromethane, and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The residue was concentrated under reduced pressure to give the crude title product 16 g (8 mg), which was used directly in the next step without purification.

MCm/z (ESI): 634.1 [M+1].

Stage 7

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

Crude 16g (8 mg) was dissolved in 1 ml of N,N-dimethylformamide. 8g (8.8 mg, 18.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (5.2 mg, 18.8 μmol) were added to the reaction solution. The reaction solution was stirred at room temperature for 30 min and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min) to give the title product 16 (1.0 mg, yield 7.2%).

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

Example 1-17

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

Stage 1

Tert-butyl-1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxatriundec-31-oate 17b

1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxadioctadecyl-28-ol 17a (0.34 g, 0.67 mmol, supplier Bide) was dissolved in 10 ml of dichloromethane, and silver oxide (0.24 g, 1.01 mmol), tert-butyl bromoacetate (0.16 g, 0.81 mmol) and potassium iodide (0.07 g, 0.40 mmol) were added successively. The reaction solution was stirred at room temperature for 3 h and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 17b (0.42 mg, yield 100%).

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

Stage 2

Tert-butyl 29-hydroxy-3,6,9,12,15,18,21,24,27-nonaxogeneicosano-1-oate 17c

17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran and palladium catalyst on carbon support (110 mg, 10% loading, dry) was added. The reaction solution was purged with hydrogen three times, stirred at 60 °C for 3 h, filtered through celite and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the crude title product 17c (357 mg), which was used directly in the next step without purification.

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

Stage 3

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

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

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

Stage 4

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

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

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

Stage 5

Tert-butyl-1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azatriundec-31-oate 17f

(1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) was dissolved in 10 mL of dichloromethane and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol) were added. The reaction solution was purged with argon three times, crude 17e (220 mg, 0.417 mmol) was added, stirred at room temperature for 1 h, 15 mL of water was added and extracted with dichloromethane (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (15 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing solvent system B to give the title product 17f (122 mg, yield 39.2%).

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

Stage 6

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

7f (122 mg, 0.163 mmol) was dissolved in 0.8 ml of dichloromethane, and 0.4 ml of trifluoroacetic acid was added thereto. The reaction solution was stirred at room temperature for 1 h, diluted with 15 ml of dichloromethane and concentrated under reduced pressure; 10 ml of n-hexane was added thereto, followed by concentration under reduced pressure; these procedures were repeated twice. Then, 10 ml of toluene was added to the reaction solution, concentrated under reduced pressure, then suspended with 10 ml of a mixed solvent of n-hexane:diethyl ether = 5:1 three times to adjust the pH to close to 7, concentrated and dried with an oil pump to give the title product 17g (98 mg, yield 86.8%).

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

Stage 7

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

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

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

Stage 8

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

17 h (39 mg, 69.6 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1 h, concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times, followed by concentration under reduced pressure. The obtained crude product was dissolved in 2 ml of N,N-dimethylformamide, and (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, obtained by the method disclosed in “Examples 7-12 on p. 13 of the description in patent application CN108853514A”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 μmol) were added. The reaction solution was stirred at room temperature for 1 h, 10 ml of water was added, and extracted with ethyl acetate (10 ml × 3). The organic phases were combined, washed with a saturated sodium chloride solution (10 ml × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 17j (48 mg, yield 83.9%).

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

Stage 9

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

17j (48 mg, 58.4 μmol) was dissolved in 1.4 ml of 3% (v/v) dichloroacetic acid in dichloromethane. The reaction solution was cooled to 0-5 °C in an ice water bath, triethylsilane (21 mg, 180.6 μmol) was added thereto, and stirred in an ice water bath for 3 h. The reaction solution was concentrated under reduced pressure in an ice water bath to remove half of the organic solvent, 5 ml of diethyl ether was added thereto, then warmed naturally to room temperature, suspended to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 17k (33 mg, yield 84.1%).

Stage 10

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

To the reaction flask was added 1b (20 mg, 42.4 μmol), followed by adding 1 ml of 10% (v/v) methanol solution in dichloromethane solution. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, one drop of triethylamine was added and stirred until 1b was dissolved. 17k (33 mg, 49.1 μmol) was dissolved in 1 ml of 10% (v/v) methanol solution in dichloromethane, then the reaction mixture was added dropwise to the above reaction solution, followed by adding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol). The reaction solution was warmed to room temperature and stirred for 1 h, 10 ml of dichloromethane and 5 ml of water were added, stirred for 5 min and kept for separation of layers. The organic phase was collected and the aqueous phase was extracted with dichloromethane (10 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 171 (37 mg, yield 80.2%).

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

Stage 11

(1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarboni (l)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 (15.5 mg, 14.23 μmol) was dissolved in 0.6 ml of dichloromethane, and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended in 3 ml of n-hexane and the upper layer of n-hexane was removed; these procedures were repeated three times. The reaction solution was concentrated under reduced pressure and then dried using an oil pump. The resulting crude product was dissolved in 1 ml of N,N-dimethylformamide. 17g (11mg, 15.92μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0mg, 21.68μmol) was added to the reaction solution, purged with nitrogen three times, stirred at room temperature for 30min, and purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5μm 19×250mm; mobile phase: A-water (10mmol _NH4OAC ), B-acetonitrile, gradient elution, flow rate: 18mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 17 (6mg, yield 27.4%).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.98 (d, 1H), 8.76 (s, 1H), 8.20 (br, 1H), 8.12-7.95 (m, 3H), 7.93-7.76 (m, 2H), 7.75-7.66 (m, 2H), 7.24 (s, 1H), 7.20-7.05 (m, 6H), 6.97 (s, 1H), 6.64 (br, 1H), 6.55 (d, 1H), 6.47 (s, 1H), 5.61-5.52 (m, 2H), 5.37 (s, 1H), 5.33-5.23 (m, 2H), 5.18 (s, 1H), 5.13 (s, 1H), 5.05 (s, 1H), 5.00 (s, 1H), 4.65-4.55 (m, 2H), 4.53-4.45 (m, 1H), 4.38-4.28 (m, 2H), 3.84 (s, 2H), 3.67 (d, 3H), 3.60-3.40 (m, 3H), 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).

Example 1-18

(1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10.13 -dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaase -tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

Stage 1

Benzyl-(R)-2-cyclopropyl-2-hydroxyacetate 18a

Benzyl-(S)-2-cyclopropyl-2-hydroxyacetate 18b

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

Stage 2

Benzyl-(R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 18с

8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), 18a (2.0 g, 9.70 mmol) was added. The reaction solution was cooled to 0-5 °C in ice water bath, potassium tert-butoxide (1.89 g, 16.84 mmol) was added, stirred in ice water bath for 10 min, ethyl acetate (30 mL) and water (20 mL) were added and kept for layer separation. The aqueous layer was extracted with chloroform (30 mL × 5), the organic layers were combined and concentrated under reduced pressure. The obtained residue was dissolved in 1,4-dioxane (32 ml) and water (8 ml), sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) were added, stirred at room temperature for 2 h, water (30 ml) was added and extracted with ethyl acetate (50 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (30 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The obtained residue was purified by column chromatography with a developing solvent system C to give the title product 18 s (1.3 g, yield 30.0%).

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

Stage 3

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

18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL) and palladium catalyst on carbon support (260 mg, 10% loading, dry) was added. The reaction solution was purged with hydrogen three times, stirred at room temperature for 5 h, filtered through celite and the filter cake was washed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to give the crude title product 18d (980 mg), which was used directly in the next step without purification.

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

Stage 4

2,4-dimethoxybenzyl(R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-ether 18e

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

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

Stage 5

2,4-dimethoxybenzyl(R)-2-((2-aminoacetylamino)methoxy)-2-cyclopropyl acetate 18f

18e (33 mg, 57.4 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 18f (21 mg), which was directly used in the next step without purification.

Stage 6

2,4-dimethoxybenzyl(11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicose-20-oate 18g

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

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

Stage 7

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

18g (37mg, 44.3μmol) was dissolved in 1.4ml of 3% (v/v) dichloroacetic acid in dichloromethane. The reaction solution was cooled to 0-5°C in an ice water bath, triethylsilane (15.4mg, 132.4μmol) was added thereto, and stirred in an ice water bath for 3h. The reaction solution was concentrated under reduced pressure in an ice water bath to remove half of the organic solvent, 5ml of diethyl ether was added thereto, then warmed naturally to room temperature, suspended to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 18h (24mg, yield 79.1%).

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

Stage 8

(9H-fluoren-9-yl)methyl((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamate 18i

To the reaction flask was added 1b (30 mg, 63.6 μmol), followed by adding 1 ml of 10% (v/v) methanol solution in dichloromethane solution. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, one drop of triethylamine was added and stirred until 1b was dissolved. 18h (65 mg, 94.8 μmol) was dissolved in 1 ml of 10% (v/v) methanol solution in dichloromethane, then the reaction mixture was added dropwise to the above reaction solution, followed by adding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol). The reaction solution was warmed to room temperature and stirred for 1 h, 10 ml of dichloromethane and 5 ml of water were added, stirred for 5 min and kept for separation of layers. The organic phase was collected and the aqueous phase was extracted with dichloromethane (10 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 18i (25 mg, yield 35.6%).

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

Stage 9

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

18i (12 mg, 10.9 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 h, concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 18j (10 mg), which was directly used in the next step without purification.

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

Stage 10

(1r,4r)-R-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10.13 -dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaase -tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

Crude 18j (10 mg) was dissolved in 1 ml of N,N-dimethylformamide. 17g (8.5 mg, 12.3 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol) were added to the reaction solution. The reaction solution was stirred at room temperature for 30 min, filtered and purified by high-performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAC), B-acetonitrile, gradient elution, flow rate: 18 ml/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 18 (9.5 mg, 56.2% yield).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.77 (d, 1H), 8.59-8.55 (m, 1H), 8.42 (d, 1H), 8.37-8.28 (m, 1H), 8.25-8.06 (m, 2H), 7.96-7.86 (m, 1H), 7.86-7.70 (m, 2H), 7.32-7.28 (m, 1H), 7.25-7.14 (m, 3H), 6.67 (m, 1H), 5.96 (s, 1H), 5.80-5.72 (m, 1H), 5.62-5.52 (m, 2H), 5.43-5.30 (m, 3H), 5.28-5.17 (m, 2H), 5.12-5.08 (m, 1H), 4.72-4.35 (m, 8H), 3.95-3.70 (m, 13H), 3.35-3.22 (m, 14H), 2.42-2.32 (m, 3H), 2.05-1.98 (m, 4H), 1.88-1.82 (m, 12H), 1.47-1.39 (m, 3H), 1.32-1.18 (m, PN), 0.90-0.80 (m, 4H), 0.52-0.37 (m, 3H), 0.32-0.18 (m, 2H).

Example 1-19

(1r,4r)-R-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10.13 -dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaase -tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

Benzyl-(S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 19a

18b (252 mg, 1.22 mmol) was added to the reaction flask followed by 4 mL of dichloromethane. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, lithium tert-butoxide (98 mg, 1.22 mmol) was added, stirred in an ice water bath for 15 min until the solution became clear, then 8b (300 mg, 814.3 μmol) was added and stirred in an ice water bath for 2.5 h. Water (10 mL) was added to separate the liquids. The aqueous phase was extracted with dichloromethane (8 ml × 2), the organic phases were combined and washed with water (10 ml × 1), washed with saturated brine (10 ml × 2), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 19a (282 mg, yield 67.2%).

Stage 2

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

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

Stage 3

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

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

Stage 4

2,4-dimethoxybenzyl(S)-2-((2-aminoacetylamino)methoxy)-2-cyclopropyl acetate 19d

19c (60 mg, 104.4 μmol) was dissolved in 1 ml of dichloromethane and 0.5 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 19d (21 mg), which was directly used in the next step without purification.

Stage 5

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

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

Stage 6

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

19e (70 mg, 83.7 μmol) was dissolved in 2.5 ml of 3% (v/v) dichloroacetic acid in dichloromethane. The reaction solution was cooled to 0-5 °C in an ice water bath, triethylsilane (29 mg, 249.4 μmol) was added thereto, and stirred in an ice water bath for 3 h. The reaction solution was concentrated under reduced pressure in an ice water bath to remove half of the organic solvent, 5 ml of diethyl ether was added thereto, then warmed naturally to room temperature, suspended to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 19f (57 mg, yield 99.2%).

Stage 7

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

To the reaction flask was added 1b (30 mg, 63.6 μmol), followed by adding 1 ml of 10% (v/v) methanol solution in dichloromethane solution. The reaction solution was purged with argon three times, cooled to 0-5 °C in an ice water bath, one drop of triethylamine was added and stirred until 1b was dissolved. 19f (57 mg, 83.1 μmol) was dissolved in 1 ml of 10% (v/v) methanol solution in dichloromethane, then the reaction mixture was added dropwise to the above reaction solution, followed by adding 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol). The reaction solution was warmed to room temperature and stirred for 1 h, 10 ml of dichloromethane and 5 ml of water were added, stirred for 5 min and kept for separation of layers. The organic phase was collected and the aqueous phase was extracted with dichloromethane (10 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 19g (56 mg, yield 79.8%).

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

Stage 8

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

19g (4?6 mg, 4.16 μmol) was dissolved in 1.5 ml of dichloromethane, and 0.75 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.6 h, concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; these procedures were repeated twice. The residue was suspended with 3 ml of n-hexane, and the upper n-hexane layer was removed; these procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 19h (4.0 mg), which was directly used in the next step without purification.

Stage 9

(1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hec sahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,3 5,38,41,44-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 19

Crude 19h (4.0 mg) was dissolved in 1 ml of N,N-dimethylformamide. 17g (2.9 mg, 4.2 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol) were added to the reaction solution. The reaction solution was stirred at room temperature for 40 min, filtered and purified by high-performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A-water (10 mmol NH ₄ OAC), B-acetonitrile, gradient elution, flow rate: 18 ml/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, 32.4% yield).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.71-8.62 (m, 1H), 8.59-8.51 (m, 1H), 8.34-8.26 (m, 1H), 8.14-8.02 (m, 2H), 7.95-7.86 (m, 1H), 7.83-7.69 (m, 2H), 7.35-7.31 (m, 1H), 7.29-7.11 (m, 3H), 7.01 (s, 1H), 6.72-6.50 (m, 3H), 5.59-5.50 (m, 2H), 5.42 (s, 2H), 5.38-5.18 (m, 3H), 4.79-4.69 (m, 2H), 4.61-4.42 (m, 3H), 3.91 (s, 2H), 3.79-3.65 (m, 4H), 3.63-3.44 (m, 13H), 3.41-3.30 (m, 2H), 3.26-3.09 (m, 5H), 3.08-2.84 (m, 4H), 2.81-2.64 (m, 3H), 2.42-2.28 (m, 3H), 2.24-2.12 (m, 2H), 2.05-1.93 (m, 4H), 1.89-1.77 (m, 2H), 1.72-1.56 (m, 3H), 1.53-1.38 (m, 3H), 1.34-1.10 (m, PN), 0.94-0.78 (m, 5H), 0.52-0.35 (m, 3H).

Example 1-20 (reference example)

The title compound 20 was synthesized by the method disclosed in Example 58 of No. 163 of Patent Specification CN104755494A.

The following antibodies can be obtained using a conventional antibody production method and can be obtained, for example, by constructing a vector, transfecting eukaryotic cells such as HEK293 cells (Life Technologies, Cat. No. 11625019), expressing and purifying.

The sequence of trastuzumab is shown below:

Light chain

Heavy chain

The sequence of pertuzumab is given below:

Light chain

Heavy chain

Below is the sequence of the B7H3 antibody 1F9DS:

Light chain

Heavy chain

Example 1-21 ADC-1

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking, and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 2.0 mL of the resulting solution was collected and subjected to reaction.

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

Average value calculated by UV-HPLC: n=5.09.

Example 1-22 ADC-2

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking, and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 2.0 mL of the resulting solution was collected and subjected to reaction.

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

Average value calculated by UV-HPLC: n=7.39.

Example 1-23 ADC-3

An aqueous solution of tris(2-carboxyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 2.0 mL of the resulting solution was collected and subjected to reaction.

Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, and the reaction mixture was added to the above 2.0 mL of the solution, placed in a water bath shaker, and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (5.24 mg/mL, 1.1 mL) of an exemplary ADC-3 product of the general formula FADC-3, and the buffer was stored at 4 °C. The average value calculated by UV-HPLC: n=7.36.

Example 1-24 ADC-4

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 3.74 mL, 13.38 mg/mL, 0.338 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking, and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg/mL, 1.3 mL of the resulting solution was collected and subjected to reaction.

Compound 9 - Compound 9-A with a shorter retention time (1.0 mg, 0.93 μmol) was dissolved in 0.10 mL of DMSO, and the reaction mixture was added to the above 1.3 mL of the solution, placed in a water bath shaker and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.72 mg/mL, 2.36 mL) of an exemplary ADC-4 product of the general formula FADC-4A, and the buffer was stored at 4 °C.

Average value calculated by UV-HPLC: n=7.39.

Example 1-25 ADC-5

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 3.0 mL, 6.70 mg/mL, 0.136 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking, and the reaction was stopped; the reaction solution was cooled to 25°C, 0.614 mL of the resulting solution was collected and subjected to reaction.

Compound 9 - Compound 9-A with a shorter retention time (0.5 mg, 0.42 μmol) was dissolved in 0.031 mL of DMSO, and the reaction mixture was added to the above 0.614 mL of the solution, placed in a shaker with a water bath and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (3.08 mg/mL, 0.82 mL) of an exemplary product ADC-5 of the general formula FADC-4A, and the buffer was stored at 4 °C.

Average value calculated by UV-HPLC: n=3.16.

Example 1-26 ADC-6

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 3.74 mL, 13.38 mg/mL, 0.338 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking, and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg/mL, 0.75 mL of the resulting solution was collected and subjected to reaction.

Compound 9 - Compound 9-B with a longer retention time (0.68 mg, 0.63 μmol) was dissolved in 0.10 mL of DMSO, and the reaction mixture was added to the above 0.75 mL of the solution, placed in a water bath shaker, and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.78 mg/mL, 1.78 mL) of an exemplary ADC-b product of the general formula FADC-4A, and the buffer was stored at 4 °C.

Average value calculated by UV-HPLC: n=3.94.

Example 1-27 ADC-7

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 5.0 mL, 10 mg/mL, 0.338 μmol) of pertuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 1.0 mL of the resulting solution was collected and subjected to reaction.

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

Average value calculated by UV-HPLC: n=6.91.

Example 1-28 ADC-8

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 5.0 mL, 10 mg/mL, 0.338 μmol) of pertuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 1.6 mL of the resulting solution was collected and subjected to reaction.

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

Average value calculated by UV-HPLC: n=6.58.

Example 1-29 ADC-9

An aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution with pH=6.5; 5.0 mL, 10 mg/mL, 0.338 μmol) of pertuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped; the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/mL, 0.8 mL of the resulting solution was collected and subjected to reaction.

Compound 9 - Compound 9-A with a shorter retention time (0.55 mg, 0.5 μmol) was dissolved in 0.1 mL of DMSO, and the reaction mixture was added to the above 0.8 mL of the solution, placed in a water bath shaker and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (2.27 mg/mL, 1.11 mL) of an exemplary product ADC-9 of the general formula FADC-9A, and the buffer was stored at 4 °C.

Average value calculated by UV-HPLC: n=3.16.

Example 1-30 ADC-10

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.574 mL, 38.78 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 14, a compound with a shorter retention time (0.64 mg, 588 nmol), was dissolved in 40 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (5.48 mg/mL, 1.03 mL) of an exemplary product ADC-10 of the general formula FADC-10, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.25.

Example 1-31 ADC-11

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.646 mL, 43.64 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

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

Average value calculated using UV-Vis detection: n=7.03.

Example 1-32 ADC-12

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

Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (3.34 mg/mL, 1.45 mL) of an exemplary product ADC-12 of the general formula FADC-12, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.93.

Example 1-33 ADC-13

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

Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (2.37 mg/mL, 0.88 mL) of an exemplary product ADC-13 of the general formula FADC-13, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.53.

Example 1-34 ADC-14

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.592 mL, 40.0 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.30 mg/mL, 12.0 mL) of an exemplary product ADC-14 of the general formula FADC-14, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.61.

Example 1-35 ADC-15

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.592 mL, 40.0 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

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

Average value calculated using UV-Vis detection: n=7.89.

Example 1-36 ADC-16

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

Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL /DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.32 mg/mL, 12.0 mL) of an exemplary product ADC-16 of the general formula FADC-16, and the buffer was stored at 4 °C.

Average value calculated using UV-Vis detection: n=7.43.

Example 1-37 ADC-17

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 2.0 mL, 135.12 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 9 - Compound 9-A with a shorter retention time (2.22 mg, 2067 nmol) was dissolved in 175 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.32 mg/mL, 12.0 mL) of an exemplary product ADC-17 of the general formula FADC-4A, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=5.42.

Example 1-38 ADC-18 (Reference Example)

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 1.5 mL, 101.3 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.79 mg/mL, 13.0 mL) of an exemplary product ADC-18 of the general formula FADC-18, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.23.

Example 1-39 ADC-19

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 1.36 mL, 91.9 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 9 - Compound 9-A with a shorter retention time (2.0 mg, 1862 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.73 mg/mL, 13.0 mL) of an exemplary product ADC-19 of the general formula FADC-4A, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.26.

Example 1-40 ADC-20

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 1.5 mL, 101.3 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 10, a compound with a longer retention time (2.0 mg, 1815 nmol), was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.73 mg/mL, 13.0 mL) of an exemplary product ADC-20 of the general formula FADC-1, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.43.

Example 1-41 ADC-21 (Reference Example)

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 1.86 mL, 125.4 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (2.91 mg/mL, 4.44 mL) of an exemplary product ADC-21 of the general formula FADC-18, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.23.

Example 1-42 ADC-22

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

Compound 9 - Compound 9-A with a shorter retention time (2.1 mg, 1955 nmol) was dissolved in 150 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (3.56 mg/mL, 3.98 mL) of an exemplary product ADC-22 of the general formula FADC-4A, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.79.

Example 1-43 ADC-23 (reference example)

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

Compound 20 (362 mg, 350 nmol) was dissolved in 7.12 μL of MeCN and 3.56 mL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaker with a water bath and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by successive desalting through ultrafiltration membranes with 2% (v/v) MeCN and 1% (v/v) DMSO-containing aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution at pH=6.5) and aqueous succinic acid buffer solution (0.01 M aqueous succinic acid buffer solution at pH=5.3), then sucrose was added to 60 mg/mL and Tween-20 to 0.2 mg/mL, and the reaction solution was lyophilized to obtain a lyophilized powder sample of an illustrative product ADC-23 of the general formula FADC-18, and the sample was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.05.

Example 1-44 ADC-24

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

Compound 9 Compound 9-A with a shorter retention time (241 mg, 224 μmol) was dissolved in 13.76 μL of MeCN and 6.88 mL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaker with a water bath and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by successive desalting through ultrafiltration membranes with 4% (v/v) MeCN and 2% (v/v) DMSO-containing aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution at pH=6.5) and aqueous succinic acid buffer solution (0.01 M aqueous succinic acid buffer solution at pH=5.3), then sucrose was added to 60 mg/mL and Tween-20 to 0.2 mg/mL, and the reaction solution was lyophilized to obtain a lyophilized powder sample of an exemplary product ADC-24 of the general formula FADC-4A, and the sample was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.07.

Example 1-45 ADC-25

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 2.14 mL, 144.60 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 9 - Compound 9-A with a shorter retention time (3.0 mg, 2793 nmol) was dissolved in 150 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.28 mg/mL, 13.0 mL) of an exemplary product ADC-25 of the general formula FADC-25, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.87.

Example 1-46 ADC-26 (Reference Example)

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.89 mL, 60.14 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.61 mg/mL, 4.0 mL) of an exemplary product ADC-26 of the general formula FADC-26, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.15.

Example 1-47 ADC-27

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.89 mL, 60.14 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 9 - Compound 9-A with a shorter retention time (1.02 mg, 950 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaker with a water bath, and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.94 mg/mL, 3.5 mL) of an exemplary product ADC-27 of the general formula FADC-25, and the buffer was stored at 4 °C.

Average value calculated using UV-Vis detection: n=6.11.

Example 1-48 ADC-28 (Reference Example)

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 2.36 mL, 159.47 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.29 mg/mL, 13.0 mL) of an exemplary product ADC-28 of the general formula FADC-26, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.46.

Example 1-49 ADC-29

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

Compound 9 - Compound 9-A with a shorter retention time (1.29 mg, 1201 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a water bath shaker, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (2.63 mg/mL, 2.4 mL) of an exemplary product ADC-29 of the general formula FADC-25, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=7.24.

Example 1-50 ADC-30 (reference example)

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.86 mL, 58.4 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.61 mg/mL, 4.0 mL) of an exemplary product ADC-30 of the general formula FADC-26, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.15.

Example 1-51 ADC-31

An aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 0.89 mL, 60.14 nmol) of anti-B7H3 antibody 1F9DS at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaking water bath, and reacted at 25°C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by desalting on a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (1.47 mg/mL, 4.5 mL) of an exemplary product ADC-31 of the general formula FADC-31, and the buffer was stored at 4°C.

Average value calculated using UV-Vis detection: n=6.33.

Analysis of drug loading in ADC stock solution Objective and principle of the experiment

The ADC stock solution is a drug with a cross-linked antibody, and its mechanism of treating diseases is to transport toxin molecules into cells depending on the targeting ability of the antibody to kill cells. The drug load plays a decisive role in the effectiveness of the drug. The drug load in the ADC stock solution was determined using the UV method. Experimental procedure

Cuvettes containing sodium succinate buffer were placed in the reference well and sample well, and the absorbance of the blank solution was subtracted. The cuvette containing the test solution was then placed in the sample cuvette, and the absorbance was determined at 280 nm and 370 nm.

Calculation of results: The loading value of the ADC stock solution was determined by UV spectrophotometry (device: Thermo nanodrop2000 UV spectrophotometer), based on the principle that the total optical density of the ADC stock solution at a certain wavelength is the sum of the optical density values of the cytotoxic drug and the monoclonal antibody at that wavelength, namely:

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

С _LS : concentration of the drug;

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

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

b: the optical path length is 1 cm.

Similarly, the equation for the total absorbance of a sample at 370 nm can be written as:

ε _LS-370 : the average molar extinction coefficient of the drug at 370 nm is 19000;

С _LS : concentration of the drug;

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

C _mab : concentration of the stock solution of monoclonal antibody trastuzumab;

b: the optical path length is 1 cm.

The drug load can be calculated using both equations (1) and (2) and the attenuation coefficients of the monoclonal antibody and drug at both wavelengths and their concentrations.

Drug load = C _LS / C _mab .

Biological Evaluation Test Example 1-1: In vitro tumor cell proliferation inhibition test by the compounds disclosed herein

I. Objective

This experiment was designed to detect the inhibitory activity of the pharmaceutical compounds of the present invention on the in vitro proliferation of U87MG cells (Cell Bank, Chinese Academy of Sciences, Cat. No. TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Cat. No. HTB-30). The cells were treated in vitro with the compound at different concentrations. After 6 days of culture, the cell proliferation was measured using CTG reagents (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat. No. G7573) and the in vitro activity of the compound was evaluated according to the IC ₅₀ value.

II. Method

The method for testing the inhibition of proliferation of U87MG cells in vitro was described below as an example of a method for analyzing the inhibitory activity of the compounds of the present invention on the proliferation of tumor cells in vitro. This method was also applicable, but not limited to, to a test for the inhibitory activity on the proliferation of other tumor cells in vitro.

1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, Cat. No. SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, Cat. No. 16600-108) containing 10% FBS, respectively.

2. Cell preparation: U87MG and SK-BR-3 cells in logarithmic growth phase were washed once with PBS (phosphate buffer, Shanghai BasalMedia Technologies Co., Ltd.) and then digested with 2-3 ml trypsin (0.25% trypsin-EDTA (1×), Gibico, Life Technologies) for 2-3 min. After the cells were completely digested, 10-15 ml culture medium was added to elute the digested cells. The mixtures were centrifuged at 1000 rpm for 5 min, and the supernatants were discarded. The cells were then resuspended in 10-20 ml culture medium to obtain single-cell suspensions.

3. Cell seeding: Each of the U87MG and SK-BR-3 single cell suspensions were mixed thoroughly and adjusted with culture medium to a cell density of 2.75 × ¹⁰³ cells/mL and 8.25 × ¹⁰³ cells/mL, respectively. The density-adjusted cell suspensions were mixed thoroughly and added to 96-well cell culture plates at 180 μL/well. Only 200 μL of medium was added to each peripheral well of the 96-well plate. The plate was incubated in an incubator for 24 h (37°C, 5% _CO2 ).

4. Preparation of compound: The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution with an initial concentration of 10 mM.

Low molecular weight compounds were prepared at an initial concentration of 500 nM as follows.

The different test samples at a concentration of 100 μM (30 μl) were added to the first column of a 96-well U-bottom plate, and 20 μl of DMSO was added to each well of the second through eleventh columns. The samples in the first column (10 μl) were added to 20 μl of DMSO in the second column, and the mixtures were mixed well. The mixtures (10 μl) were added to the third column, and so on up to the tenth column. The drugs in the plate (5 μl per well) were transferred to EMEM medium (95 μl), and the mixtures were mixed well for subsequent use.

ADC was prepared at a stock concentration of 10 nM or 500 nM as follows.

The different samples to be tested at a concentration of 100 nM or 5 μM (100 μl) were added to the first column of a 96-well plate, and 100 μl of PBS was added to each well of the second through eleventh columns. The samples in the first column (50 μl) were added to 100 μl of PBS in the second column, and the mixtures were mixed well. The mixtures (50 μl) were added to the third column, and so on, by 3-fold dilutions, up to the tenth column.

5. Sample addition: The test samples prepared at different concentrations (20 µl) were added to the culture plate, using two parallel wells for each sample. The plate was incubated in an incubator for 6 days (37°C, 5% CO ₂ ).

6. Color development: The 96-well cell culture plate was removed and 90 μl of CTG solution was added to each well, followed by incubation for 10 min at room temperature.

7. Plate reading: The 96-well cell culture plate was removed and tested in a microplate reader (BMG labtech, PHERAstar FS) to determine chemiluminescence.

III. Data Analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results of the experiment are presented below in Table 1.

Conclusion: The low molecular weight fragments according to the present invention have significant inhibitory activity against the proliferation of SK-BR-3 cells and U87 cells, and the chiral centers have a certain effect on the inhibitory activity of the compounds.

Test Example 1-2: In vitro test for inhibition of tumor cell proliferation by the antibody-drug conjugates of the present invention targeting HER2

The aim of this experiment was to detect the inhibitory activity of the HER2-targeted antibody-drug conjugates of the present invention against in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, Cat. No. HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, Cat. No. HTB-132). The cells were treated in vitro with the compound at different concentrations. After 6 days of culture, cell proliferation was measured using CTG reagents and the in vitro activity of the compound was evaluated according to the IC ₅₀ value.

According to the assay procedure of Test Example 1, the test cells were SK-BR-3 and MDA-MB-468, and the cell culture medium was McCoy's 5A medium containing 10% FBS (Gibco, Cat. No. 16600-108), EMEM medium containing 10% FBS (GE, Cat. No. SH30024.01), and L-15 medium containing 10% FBS (ThermoFisher, Cat. No. 11415-114), respectively. The cell density of the three cell strains was adjusted to 8.33 × ¹⁰³ cells/mL, 8.33 × ¹⁰³ cells/mL, and 1.39 × ¹⁰⁴ cells/mL with the culture medium, respectively, and the cell suspensions after density adjustment were mixed well and added to 96-well cell culture plates at 180 μL/well. The test results of the respective compounds are shown in Table 2 below.

Conclusion: The antibody-drug conjugates targeting HER2 targets according to the present invention have significant inhibitory activity against HER2-positive SK-BR-3 cells; however, the proliferation inhibitory activity of the compounds against HER2-negative MDA-MB-468 cells is weak; the compounds have good selectivity.

Test Case 1-3: Her2-ADC Plasma Stability Test

ADC-19, ADC-18, and ADC-20 samples, human plasma, monkey plasma (Shanghai Medicilon Inc.), and 1% BSA (Sigma) in PBS (Sangon Biotech (Shanghai)) were filtered through a 0.22 μm filter for sterilization. ADC-19, ADC-18, and ADC-20 were added to sterile plasma or 1% BSA in PBS, respectively, at a final concentration of 200 μg/mL, and the reaction solution was incubated in an incubator at 37°C; the day of incubation was marked as day 0, and samples were recovered at days 7, 14, and 21, respectively, for free toxin detection.

25 μl of samples were extracted into a 96-well plate; 50 μl of the internal standard working solution (100 ng/ml camptothecin solution in acetonitrile) and 150 μl of acetonitrile were added; the mixture was shaken for 5 min and centrifuged for 10 min (4000 rpm), 5 μl of the resulting samples were used for LC/MS/MS analysis (Applied Biosystems, USA).

The results showed that ADC-19 was reasonably stable in both human and monkey plasma, as well as in a 1% BSA solution in PBS, with a free toxin release rate of no more than 2.1% at the highest level, and was generally stable at day 14, see FIG. 1A.

ADC-18 was poorly stable in human and monkey plasma with maximum free toxin release rates of 14.5% and 8.10%, respectively. It was relatively stable in a 1% BSA solution in PBS, see FIG. 1B.

ADC-20 was poorly stable in human plasma, monkey plasma, and 1% BSA in PBS, with maximum free toxin release rates of 21.7%, 29.7%, and 21.7%, respectively. It was degraded in 1% BSA in PBS, see FIG. 1C.

Test Case 1-4: Evaluation of Drug Efficacy in LMT-1 Tumor-Bearing Mice

1. Purpose

Nude nu/nu mice were used as experimental animals, and the therapeutic effect of Her2-ADC antibodies T-DM1, ADC-21 and ADC-24 was assessed in nude mice bearing xenograft of trastuzumab (Herceptin) drug-resistant human breast cancer LMT-1 cells after intraperitoneal injection.

2. Investigational medicinal products and materials 2-1. Investigational medicinal products

T-DM1 (obtained according to US20050169933)

ADC-21: 3 mg/kg ADC-21: 10 mg/kg ADC-24: 3 mg/kg

ADC-24: 10 mg/kg

Blank control (blank): PBS

2-2. Preparation method: all were diluted with PBS.

2-3. Experimental animals

Nu/nu nude mice purchased from Beijing Vital River.

3. Method of analysis

Mice were subcutaneously inoculated in the right flank with LMT-1 cells (Nanjing Kebai) (5× ¹⁰⁶ /mouse with 50% artificial basement membrane), and the tumors were grown for 8 days to 203.09±11.94 mm, after which the animals were randomly divided into groups (d1), 8 per group, a total of 6 groups.

The drug was administered via intraperitoneal injection a total of 2 times. Tumor volumes and body weights were measured and recorded twice a week.

Excel 2003 statistical analysis software was used. Mean values were calculated as avg; SD values were calculated as STDEV; SEM values were calculated as STDEV/SQRT; and P-value of between-group difference was calculated as TTEST.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative volume (RTV)=V _t /V ₀

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

Where is V₀and V_T- tumor volume at the beginning and end of the experiment, respectively. C_RTV and T_RTV- relative tumor volumes in the blank group (vehicle, PBS) and in the experimental groups, respectively, at the end of the experiment. 4. Experimental results

The experimental results are shown in FIG. 2. The experiment was terminated after two intraperitoneal injections and then observed for 34 days. T-DM1 (10 mg/kg) had no tumor inhibitory effect; 3 mg/kg ADC-21 had a tumor inhibition rate of 46.22% (P<0.01); 10 mg/kg ADC-21 had a tumor inhibition rate of 56.77% (P<0.001); 3 mg/kg ADC-24 had a tumor inhibition rate of 62.77% (P<0.001); 10 mg/kg ADC-24 had a tumor inhibition rate of 76.32% (P<0.001). At the same dosage, the tumor inhibition effect of ADC-24 was significantly better than that of ADC-21.

Test Case 1-5: Evaluation of Drug Efficacy in SK-BR-3 Tumor-Bearing Mice

1. Purpose

Nude nu/nu mice were used as experimental animals, and the therapeutic effect of Her2-ADC antibodies ADC-21 and ADC-22 was evaluated in nude mice bearing human breast cancer cell xenograft SK-BR-3 after intraperitoneal injection.

2. Investigational medicinal products and materials 2-1.

Investigational medicinal products

ADC-21: 1 mg/kg

ADC-21: 6 mg/kg

ADC-22: 1 mg/kg

ADC-22: 6 mg/kg

Blank control (blank): PBS.

2-2. Preparation method: all were diluted with PBS.

2-3. Experimental animals

Nu/nu nude mice purchased from Beijing Vital River.

3. Method of analysis

Mice were subcutaneously inoculated in the right flank with SK-BR-3 (ATCC) cells (5× ¹⁰⁶ /mouse with 50% artificial basement membrane), and tumors were grown for 20 days to 153.34±11.73 ^mm3 , after which the animals were randomly divided into groups (d 0), 8 per group, a total of 5 groups.

The drug was administered via intraperitoneal injection once in total. Tumor volumes and body weights were measured twice a week and the results were recorded.

Excel 2003 statistical analysis software was used. Mean values were calculated as avg; SD values were calculated as STDEV; SEM values were calculated as STDEV/SQRT; and P-value of between-group difference was calculated as TTEST.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative volume (RTV)=V _T /V ₀

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

Where V ₀ and V _T are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes in the control blank group and in the experimental groups, respectively, at the end of the experiment.

4. Experimental results

The experimental results are shown in FIG. 3. The experiment was terminated after one intraperitoneal injection, and then observed for 28 days. 1 mg/kg ADC-21 had a tumor inhibition rate of 15.01%; 6 mg/kg ADC-21 had a tumor inhibition rate of 77.4%, which was significantly different from the blank control (P<0.001). 1 mg/kg ADC-22 had a tumor inhibition rate of 19.82%; 6 mg/kg ADC-22 had a tumor inhibition rate of 98.38% (P<0.001). At the same dosage of 6 mg/kg, the tumor inhibition effect of ADC-22 was significantly better than that of ADC-21.

Test example 1-6:

Stability in plasma

ADC-25 sample was uniformly mixed with human plasma, monkey plasma and 1% BSA solution in PBS to a final concentration of 100 μg/mL, the mixture was filtered for sterilization and then incubated in a water bath at 37°C; the day of incubation was marked as day 0, and samples were recovered at days 7, 14 and 21, respectively, to detect free toxin.

Samples were removed at different time points, then placed at room temperature, shaken and mixed well; 25 μl of samples were removed per 96-well plate; 50 μl of internal standard working solution (100 ng/ml camptothecin solution in acetonitrile) and 150 μl of acetonitrile were added; the mixture was shaken for 5 min and centrifuged for 10 min (4000 rpm), 5 μl of supernatant was recovered for LC/MS/MS analysis.

The results showed the following: ADC-25 was reasonably stable in both human and monkey plasma, as well as in a solution of 1% BSA in PBS, with a free toxin release rate of no more than 2% at the highest level and was generally stable at day 14, see FIG. 4.

Test Case 1-7: Evaluation of the Therapeutic Effect of ADC on Human Brain Astrocytoma Xenograft U87MG in Nude Mice

1. Purpose

In this experiment, BALB/cA nude mice were used as experimental animals and the therapeutic effect of the ADC compounds disclosed herein on human brain astrocytoma xenograft U87MG in nude mice was evaluated.

2. Study drugs and materials 2-1. Study drugs ADC-27 (3 mg/kg)

ADC-26 (3 mg/kg)

Blank control (blank): PBS buffer at pH 7.4.

2-2. Method of preparation: PBS buffer at pH 7.4.

2-3. Experimental animals

BALB/cA nude mice: purchased from Shanghai Jiesijie Experimental Animal Company.

3. Method of analysis

In the experiment, 6-7 week-old female BALB/cA nude mice were subcutaneously inoculated with human brain astrocytoma U87MG cells (human brain astrocytoma, cell bank, Chinese Academy of Sciences, cat. No. TCHu138). On the tenth day after cell inoculation, the animals were randomly divided into groups (D 0), 8 animals per group, intraperitoneally injected once a week for a total of 3 times, and the tumor volume and body weight were measured 2-3 times a week and the data were recorded. The tumor volume (V) was calculated as follows:

V=1/2 × a × b ²

where a and b represent the length and width, respectively.

Relative volume (RTV)=V _T /V ₀

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

Where V ₀ and V _T are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes in the control blank group (blank) and in the experimental groups, respectively, at the end of the experiment. 4. Experimental Results

Intraperitoneal injection (i.p.) was performed once a week for a total of 3 times; after observation for 22 days, 3 mg/kg ADC-27 had a tumor inhibition rate of 63.3% (P<0.0001), and 3 mg/kg ADC-26 had an inhibition rate of 49.1%. ADC-27 showed more potent antitumor effect than ADC-26.

The body weight of all animals in each group was normal during the administration process, and the ADCs had no obvious toxicity or side effects. The detection results are shown in Table 3 and FIG. 5. The detected antibodies could effectively inhibit the growth of U87MG xenograft tumor in tumor-bearing nude mice and showed a dose-dependent effect.

Test Case 1-8: Evaluation of the Therapeutic Effect of ADC on Detroit 562 Human Pharyngeal Cancer Xenograft Metastasizing to Pleural Effusion in Nude Mice

1. Purpose

In this experiment, BALB/cA nude mice were used as experimental animals and the therapeutic effect of the ADC compounds disclosed herein on a xenograft of human pharyngeal cancer Detroit 562 cells metastasizing to pleural effusion in nude mice was evaluated.

2. Investigational medicinal products and materials 2-1.

Investigational drugs ADC-29 (3 mg/kg)

ADC-28 (3 mg/kg)

Negative control ADC (3 mg/kg): antibody-drug conjugate formed by coupling non-B7H3-targeted antibodies to compound 20.

2-2. Preparation method: all were diluted with PBS.

2-3. Experimental animals

BALB/cA nude mice: purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

3. Method of analysis

In the experiment, female 6-7 week-old BALB/cA nude mice were inoculated subcutaneously with Detroit 562 cells, a human pharyngeal carcinoma metastasizing to pleural effusion (ATCC, Cat. No. ATCC® CCL-138™). On the tenth day after cell inoculation, the animals were randomly divided into groups (D 0), 8 animals per group, intraperitoneally injected once a week for a total of 3 times, and tumor volume and body weight were measured 2-3 times per week and the data were recorded. Tumor volume (V) was calculated as follows:

V=1/2 × a × b ²

where a and b represent the length and width, respectively.

Relative volume (RTV)=V _T /V ₀

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

Where V ₀ and V _T are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes in the control group (negative control) and in the experimental groups, respectively, at the end of the experiment.

4. Experimental results

Intraperitoneal injection was performed once a week for a total of 3 times; after observation for 28 days, 3 mg/kg ADC-29 (3 mg/kg) had a tumor inhibition rate of 72.27% (P<0.001), and 3 mg/kg ADC-28 (3 mg/kg) had an inhibition rate of 56.2% (P<0.001). ADC-29 showed more potent antitumor effect than ADC-28.

The body weight of all animals in each group was normal during the administration process, and the ADCs had no obvious toxicity or side effects. The detection results are shown in Table 4 and FIG. 6. The detected antibodies could effectively inhibit the growth of Detroit 562 xenograft tumor in tumor-bearing nude mice and showed a dose-dependent effect.

Test Case 1-9: Evaluation of Drug Efficacy in Mice with U87-MG Tumors

1. Purpose

Balb/c nude mice were used as experimental animals, and the therapeutic effect of the anti-B7H3 antibody-drug conjugate was evaluated after intraperitoneal injection in a mouse U87MG human glioblastoma cell xenograft model.

2. Investigational medicinal products and materials 2-1.

Investigational drugs ADC-30 1 mg/kg

ADC-30 3 mg/kg ADC-31 1 mg/kg ADC-31 3 mg/kg

Blank control (blank): PBS

2-2. Preparation method: all were diluted with PBS.

2-3. Experimental animals

BALB/cA nude mice: purchased from Shanghai Slac Laboratory Animal Co., Ltd.

3. Method of analysis

Mice were subcutaneously inoculated in the right flank with U87MG cells (human brain astrocytoma, Cell Bank, Chinese Academy of Sciences, Cat. No. TCHu138) (2.5× ¹⁰⁶ /mouse), and the tumors were grown for 14 days to 167.49 ^mm3 , after which the animals were randomly divided into groups (d 1), 8 per group, a total of 5 groups.

Intraperitoneal injection was performed once a week for a total of 3 times. Tumor volumes and body weights were measured twice a week and the results were recorded.

Excel 2003 statistical analysis software was used. Mean values were calculated as avg; SD values were calculated as STDEV; SEM values were calculated as STDEV/SQRT; and P-value of between-group difference was calculated as TTEST.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative volume (RTV)=V _T /V ₀

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

Where V ₀ and V _T are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes in the control blank group (carrier) and in the experimental groups, respectively, at the end of the experiment.

4. Experimental results

The experimental results are shown in FIG. 7. Intraperitoneal injection was performed once a week for a total of 3 times; after observation for 18 days, the studied ADCs had the following tumor inhibition rates: 1 mg/kg ADC-30 had a tumor inhibition rate of 0.31%; 3 mg/kg ADC-30 had a tumor inhibition rate of 45.23% (P<0.0001); 1 mg/kg ADC-31 had a tumor inhibition rate of 39.22% (P<0.01); 3 mg/kg ADC-31 had a tumor inhibition rate of 80.24% (P<0.0001). At the same dosage, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

II. Optimization of the production process The illustrative products of the following examples have the structure shown below:

Example 2-1

An antibody stock solution (0.0004251 mmol, trastuzumab antibody diluted in 20 mM histidine-hydrochloric acid buffer to a final antibody concentration of 15 mg/mL) containing 61.71 mg trastuzumab was reacted with 0.7311 mg tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.002550 mmol) in 20 mM histidine-hydrochloric acid buffer (pH 5.6) containing 2.5 mM EDTA, with stirring in a constant temperature water bath at 0°C for 3 h to yield a solution of intermediate I.

Compound 9 - Compound 9-A with a shorter retention time (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL DMSO to obtain a solution of Compound 9-A in DMSO. To the above solution of Intermediate I was added 0.2052 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-1 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.45.

Example 2-2

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

Compound 9 - Compound 9-A with a shorter retention time (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL DMSO to obtain a solution of Compound 9-A in DMSO. To the above solution of Intermediate I was added 0.2052 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-2 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.49.

Example 2-3

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

Compound 9 Compound 9-A with a shorter retention time (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL DMSO to obtain a DMSO solution of Compound 9-A. To the above solution of Intermediate I was added 0.2052 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-3 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.39.

Example 2-4

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

Compound 9 - Compound 9-A with a shorter retention time (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL DMSO to obtain a DMSO solution of Compound 9-A. To the above solution of Intermediate I was added 0.2052 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-4 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.44.

Example 2-5

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

Compound 9 - Compound 9-A with a shorter retention time (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL DMSO to obtain a solution of Compound 9-A in DMSO. To the above solution of Intermediate I was added 0.2052 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-5 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.46.

Example 2-6

An antibody stock solution (0.0003790 mmol, trastuzumab antibody diluted with 50 mM PBS buffer to a final antibody concentration of 15 mg/mL) containing 55.02 mg trastuzumab was reacted with 0.4563 mg tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001592 mmol) in 50 mM PBS buffer (pH 6.5) containing 2.5 mM EDTA, with stirring in a constant temperature water bath at 13°C for 3 h to obtain a solution of intermediate compound I.

Compound 9 Compound 9-A with a shorter retention time (2.850 mg, 0.002653 mmol) was dissolved in 0.1839 mL DMSO to obtain a solution of Compound 9-A in DMSO. To the above solution of Intermediate I was added 0.1829 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-6 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.39.

Example 2-7

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

Compound 9 - Compound 9-A with a shorter retention time (2.850 mg, 0.002653 mmol) was dissolved in 0.1839 mL DMSO to obtain a DMSO solution of Compound 9-A. To the above solution of Intermediate I was added 0.1829 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-7 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.50.

Example 2-8

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

Compound 9 Compound 9-A with a shorter retention time (2.850 mg, 0.002653 mmol) was dissolved in 0.1839 mL DMSO to obtain a solution of Compound 9-A in DMSO. To the above solution of Intermediate I was added 0.1829 mL DMSO and to the resulting solution was added the above solution of Compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and quenched with excess cysteine. An illustrative product ADC-2-8 of the general formula FADC-4A was obtained.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.47.

Example 2-9

A stock solution of antibody (0.88 mmol, trastuzumab antibody diluted in 20 mM histidine-hydrochloric acid buffer to a final antibody concentration of 15 mg/mL) containing 127.4 g of trastuzumab was reacted with 0.83 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 2.90 mmol) in 20 mM histidine-hydrochloric acid buffer (pH 5.6) containing 2.5 mM EDTA, with stirring in a constant temperature water bath at 25°C for 3 h to yield a solution of intermediate I.

Compound 9 Compound 9-A with a shorter retention time (6.6 g, 6.14 mmol) was dissolved in 0.43 L of DMSO to obtain a DMSO solution of compound 9-A. To the above solution of intermediate compound I was added 0.43 L of DMSO, and to the resulting solution was added the above solution of compound 9-A in DMSO, which was stirred in a water bath at 25 °C for 1 h and the reaction was stopped.

The above reaction solution was purified on a Capto S Impact (GE) cation chromatography column and washed with at least 9 column volumes of 0.05 M acetate buffer containing 10% (v/v) DMSO (pH 5.5) and 6 column volumes of 0.05 M acetate buffer (pH 5.5), followed by elution with 0.05 M acetate buffer (pH 5.5, containing 0.39 M sodium chloride) to remove free toxins and residual solvent from the reaction solution. The eluate from cation chromatography was subjected to 7-fold ultrafiltration with an equal volume (30 kDa ultrafiltration membrane pack) and buffer exchange with 0.01 M succinic acid buffer (pH 5.0) at 25 °C to obtain an illustrative product ADC-A1 of the general formula FADC-4A. Samples of 4 batches were obtained as described above.

The drug load of the samples of the 4 batches, determined by reversed-phase chromatography with tandem mass spectrometry, was 5.7. The yields of the four batches were 100.8%, 98.9%, 97.4% and 99.0%, respectively.

Example 2-10

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 3.55 mL, 35.5 μmol) was added to an aqueous PBS buffer solution (0.05 M aqueous PBS buffer solution pH 6.5; 10.0 mg/mL, 164 mL, 11.08 μmol) of trastuzumab antibody at 37°C, and the reaction mixture was placed in a water bath shaker to carry out the reaction at 37°C for 3.5 h with shaking and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

Compound 9 - Compound 9-A with a shorter retention time (185 mg, 172 μmol) was dissolved in 3.88 μL of acetonitrile and 1.94 mL of DMSO, and the reaction mixture was added to the above reaction solution, placed in a shaker with a water bath and reacted at 25 °C for 3 h with shaking, and the reaction was stopped. The reaction solution was purified by successive desalting through ultrafiltration membranes with an aqueous PBS buffer solution containing 2% (v/v) acetonitrile and 1% (v/v) DMSO (0.05 M aqueous PBS buffer solution at pH 6.5) and an aqueous succinic acid buffer solution (0.01 M aqueous succinic acid buffer solution at pH 5.3) to remove small molecules, and a sample of an illustrative product ADC-B14 of the general formula FADC-4A was obtained, which was stored at 4°C.

Mean values were calculated by reversed-phase chromatography with tandem mass spectrometry: n=5.3.

Test Case 2-1: Drug Load Distribution Test

1. Method for determining RP-DAR

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

UHPLC System: UHPLC system with Waters H-Class Ultra High Performance Liquid Chromatograph

Detector: TUV detector (measurement wavelength: 280nm)

Chromatography column: Waters ACQUITY UPLC Protein BEH C4 (2.1 mm x 150 mm, 1.7 µm)

Column temperature: 80°C

Flow rate: 0.3 ml/min

Sample chamber temperature: 20°C

Registration time: 25 min

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

Mobile phase B: 0.1% DFA solution in acetonitrile

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

Injection volume: 1.0 µl

1.2. Data analysis

In the case of the drug-bound light chain (single-drug-bound light chain: _L1 ) and the drug-bound heavy chains (single-drug-bound heavy chain: _H1 , two-drug-bound heavy chains: _H2 , three-drug-bound heavy chains: _H3 , and four-drug-bound heavy chains: _H4 ), the hydrophobicity increased proportionally to the number of drug-bound drugs and the retention time increased compared with the light chain ( _L0 ) and heavy chain ( _H0 ) of the non-drug-bound antibody. Therefore, elution was performed in the following order: _L0 , _L1 , _H0 , _H1 , _H2 , _H3 , and _H4 .

Since the drug linkers absorbed UV radiation, the resulting peak area was corrected according to the amount of bound drugs using the molar absorption coefficients of the drug light chains, heavy chains and linkers according to the following expression. The calculation formula was as follows:

Light chain (ε LC-280)/(ε LC-280 + number of related drugs × ε LC-280)

Heavy chain (ε HC-280)/(ε HC-280 + number of bound drugs × ε LS-280)

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

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

ε LS-280: molar extinction coefficient of the toxin at 280 nm.

Total adjusted peak area HC = adjusted peak area H ₀ + adjusted peak area H ₁ + adjusted peak area H ₂ + adjusted peak area H ₃ + adjusted peak area H ₄

Drug loading n=2 × Σ(number of bound drugs × percentage of adjusted peak area)/100

2. Measurement results

The results showed that for the samples prepared using the same buffer system and different reconstitution reaction temperatures, the uniformity of drug load distribution in the samples was significantly improved along with the decrease in the reconstitution reaction temperature; for the samples prepared using the same reconstitution reaction temperature and different buffer systems, the distribution of drug load in the samples where the histidine-hydrochloric acid buffer system was used was more uniform.

Test Example 2-2: Free Toxin Test 1.

Method for determining free toxin

1.1. HPLC analysis was performed under the following measurement conditions:

HPLC System: UHPLC system with Waters H-Class Ultra High Performance Liquid Chromatograph

Detector: TUV detector (measurement wavelength: 370nm)

Chromatographic column: Waters ACQUITY UPLC Petide VEN C18 (130 , 2.1 mm × 150 mm, 1.7 µm)

Column temperature: 40°C

Flow rate: 0.3 ml/min

Sample chamber temperature: 10°C

Mobile phase A: 0.1% aqueous solution of trifluoroacetic acid (TFA)

Mobile phase B: 0.1% TFA solution in acetonitrile

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

Injection volume: 5.0 µl

1.2. Data analysis

The LOD limit for the toxin was calculated as follows:

Toxin limit (ppm)=0.1 × 4 × 1000/C

Note:

a) 0.1 is the concentration of the LOD toxin solution (μg/mL), 4 is the dilution factor during sample pre-treatment, 1000 is the unit conversion factor, and C is the protein concentration (mg/mL) in the sample to be measured.

b) If the peak area of the free toxin in the sample was less than the peak area of the LOD solution, it was considered to be below the limit or undetectable.

2. Measurement results

Free toxin detection was performed on ADC-A1 (batches 1-4) and ADC-B14, and the results (see Table 7) showed that cation column chromatography was not only suitable for large-scale production but also significantly reduced the free toxin content.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI PHARMACEUTICALS CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> METHOD FOR PRODUCING ANTIBODY-DRUG CONJUGATE

<130> 721020CPCT

<150>CN202010219311.2

<151> 2020-03-25

<150>CN202110297397.5

<151> 2021-03-19

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

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Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

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Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

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Phe Asn Arg Gly Glu Cys

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

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Gly Lys

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

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Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

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Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

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Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

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Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

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Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

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Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

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Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

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Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

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Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

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<221> CHAIN

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Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

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Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

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Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

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Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

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Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<---

Claims (69)

1. A method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the general formula (Pc-L _a -YD): ; where W is C _1-8 alkyl or C _1-8 alkyl-C _3-7 cycloalkyl; L ² is a chemical bond; L ³ is a peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; R ¹ is C _3-7 cycloalkyl; R ² is hydrogen; or R ¹ and R ² together with the carbon atoms bound to them form C _3-7 cycloalkyl; R ⁵ is hydrogen; R ⁶ and R ⁷ represent hydrogen; m is 0 or 1; n is a decimal or integer number between 3 and 8; Pc is an antibody or its antigen-binding fragment; where the method of obtaining includes the following stages: step (a): reacting said antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of from 1°C to 36°C; and step (b): reacting the product of step (a) with a compound of the following formula (La-Y-D): where W, L ² , L ³ , R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and m are as defined above. 2. The method for producing an antibody-drug conjugate according to claim 1, wherein the temperature conditions of the reaction in step (a) are from 4°C to 30°C, from 20°C to 30°C, and more preferably 25°C. 3. A method for producing an antibody-drug conjugate according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of 4.5 to 6.5, preferably the reaction is carried out at a pH of 5.0 to 6.0, more preferably the reaction is carried out at a pH of 5.6. 4. A method for producing 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 the buffer is selected from the group consisting of histidine salt-based buffers, phosphate buffers and acetate buffers; more preferably the buffer is an EDTA (ethylenediaminetetraacetic acid)-containing histidine salt-based buffer. 5. A method for producing an antibody-drug conjugate according to any one of claims 1 to 4, wherein the reducing agent in step (a) is selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, 1,4-dimercaptothreitol and β-mercaptoethanol, and preferably tris(2-carboxyethyl)phosphine hydrochloride. 6. A method for producing an antibody-drug conjugate according to any one of claims 1 to 5, wherein the method of production further comprises step (c), comprising purifying the product of step (b) using cationic column chromatography or affinity column chromatography; preferably step (c) comprises subjecting the product of step (b) to cationic column chromatography using a filler selected from the group consisting of Capto S Impact and Poros XS, and preferably Capto S Impact. 7. A method for producing an antibody-drug conjugate according to any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of an antibody to HER2 (ErbB2), an antibody to EGFR, an antibody to B7-H3, an antibody to c-Met, an antibody to HER3 (ErbB3), an antibody to HER4 (ErbB4), an antibody to CD20, an antibody to CD22, an antibody to CD30, an antibody to CD33, an antibody to CD44, an antibody to CD56, an antibody to CD70, an antibody to CD73, an antibody to CD105, an antibody to CEA, an antibody to A33, an antibody to Cripto, an antibody to EphA2, an antibody to G250, an antibody to MUCl, an antibody to the LeY antigen, an antibody to VEGFR, an antibody to GPNMB, an antibody to integrin, an antibody to PSMA, an antibody to tenascin-C, an antibody to SLC44A4, antibodies to mesothelin and their antigen-binding fragment; Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of trastuzumab, pertuzumab, nimotuzumab, enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, glematumamab and an antigen-binding fragment thereof. 8. A method for producing an antibody-drug conjugate according to any one of claims 1-7, wherein the antibody-drug conjugate has a structure represented by the following formula: where n is a decimal or integer number from 4 to 8. 9. A method for producing 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, and more preferably a decimal or integer from 5.3 to 6.1. 10. The method for producing an antibody-drug conjugate according to any one of claims 1 to 9, wherein the distribution of drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less; Preferably, the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to four drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains binding to one drug is 65% or more. 11. A method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the following formula: where n is a decimal or integer number from 4 to 8; where the method of obtaining includes the following stages: step (a): reacting trastuzumab with TCEP at a reaction temperature of 4°C to 30°C and a pH of 5.0 to 6.0; and step (b): reacting the product of step (a) with a compound of the following formula: 12. A method for producing an antibody-drug conjugate, wherein the antibody-drug conjugate has a structure represented by the following formula: where n is a decimal number or an integer from 4 to 8; where the method of obtaining includes the following stages: step (a): reacting trastuzumab with TCEP at a reaction temperature of 25°C and pH 5.6, where the reaction is carried out in an EDTA-containing buffer based on histidine and hydrochloric acid; step (b): reacting the product of step (a) with a compound of the following formula: step (c): subjecting the product of step (b) to purification through a cation chromatographic column. 13. An antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by the general formula (Pc-L _a -YD): ; where W is C _1-8 alkyl or C _1-8 alkyl-C _3-7 cycloalkyl; L ² is a chemical bond; L ³ is a peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; R ¹ is C _3-7 cycloalkyl; R ² is hydrogen; or R ¹ and R ² together with the carbon atoms bound to them form C _3-7 cycloalkyl; R ⁵ is hydrogen; R ⁶ and R ⁷ represent hydrogen; m is 0 or 1; n is a decimal or integer number between 4 and 8; Pc is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, antibodies to SLC44A4, antibodies to mesothelin and their antigen-binding fragment; and the distribution of the drug load in the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less. 14. The antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to four drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains binding to one drug is 65% or more. 15. An antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is obtained by the method for producing an antibody-drug conjugate according to any one of claims 1 to 9, and the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains that bind to 4 drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to drugs is 5% or less, wherein the antibody or antigen-binding fragment thereof in the antibody-drug conjugate is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, antibodies to SLC44A4, antibodies to mesothelin and their antigen-binding fragment. 16. The antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 15, wherein the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to four drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains binding to one drug is 65% or more. 17. An antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure represented by the following formula: where n is a decimal or integer number from 4 to 8; the antibody-drug conjugate is obtained by the method for producing the antibody-drug conjugate according to claim 11 or 12, and the distribution of the drug load in the antibody-drug conjugate is as follows: in the population of antibody heavy chains, the proportion of the antibody heavy chains that bind to four drugs is 4% or less, and the proportion of the antibody heavy chains that do not bind to drugs is 5% or less. 18. The antitumor antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 17, wherein the distribution of the drug load in the antibody-drug conjugate is as follows: in a population of antibody heavy chains, the proportion of antibody heavy chains binding to four drugs is 4% or less, and the proportion of antibody heavy chains not binding to drugs is 5% or less; and in a population of antibody light chains, the proportion of antibody light chains binding to one drug is 65% or more.