RU2840147C1 - Pharmaceutical composition comprising antibody-drug conjugate, and use thereof - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical preparations containing an antibody-drug conjugate, and use thereof as an anticancer drug. Disclosed are pharmaceutical compositions for the treatment of cancer associated with the expression of HER2, containing from 10 mg/ml to 30 mg/ml of an antibody-drug conjugate of the following structure:

where n is a decimal or integer from 1 to 10, polysorbate 80 at concentration of 0.05 mg/ml to 0.5 mg/ml, sucrose at concentration of 60 mg/ml to 90 mg/ml and succinic acid-sodium succinate buffer at concentration of 5 mM to 20 mM. Pharmaceutical compositions have pH from 4.5 to 5.2. Invention also discloses a method of producing a lyophilised preparation and a method of treating cancer associated with expression of HER2.

EFFECT: invention enables to obtain a stable conjugate preparation in which there are no significant changes during storage at temperatures of 2–8 °C and 25 °C for at least 3 months.

12 cl, 8 dwg, 16 tbl, 69 ex

Description

This application claims priority from Chinese Patent Application (Application No. CN202010219601.7) filed on March 25, 2020 and Chinese Patent Application (Application No. CN202110287012.7) filed on March 17, 2021.

AREA OF TECHNOLOGY

The present invention relates to the field of pharmaceuticals and, in particular, relates to a pharmaceutical composition containing a conjugate of an antibody and a drug and its use as an anticancer drug.

PRIOR ART

The information set forth herein is intended to provide only general information relating to the present invention and may not necessarily constitute prior art.

Antibody drug conjugate (ADC) links a monoclonal antibody or antibody fragment to a biologically active cytotoxin through a stable chemical linker compound, fully utilizing the binding specificity of the antibody to the surface antigens of normal cells and tumor cells and the high potency of the cytotoxin, and eliminating the disadvantage of the former that it has poor therapeutic effect, the disadvantage of the latter that it has serious toxic side effects, etc. This means that the antibody drug conjugate can bind to tumor cells more precisely and has a reduced effect on normal cells compared with conventional chemotherapeutic drugs in the past (Mullard 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 FDA in 2000 for the treatment of acute myelocytic leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).

^Adcetris® (brentuximab vedotin, Seattle Genetics) was approved under an accelerated designation 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; US7090843A; US7659241; WO2008025020). ^Adcetris® is a novel ADC-directed drug that may allow the drug to directly target CD30 on lymphoma cells and then undergo endocytosis to induce tumor cell apoptosis.

Both Mylotarg ^® and Adcetris ^® are therapies that target hematological malignancies, which are relatively simple in tissue structure compared to solid tumors. Kadcyla ^® (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA in February 2013 for the treatment of patients with HER2-positive advanced or metastatic breast cancer who are drug-resistant to tratuzumab (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 for antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor activity by inhibiting topoisomerase I. In WO2014057687; Clinical Cancer Research (2016) 22(20): 5097–5108; and Cancer Sci (2016) 107: 1039–1046 reported that camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) was applied to antibody drug conjugates (ADCs). There is still a need for further development of ADC-drugs with better therapeutic effects.

However, ADCs have more complex heterostructures than antibodies, and therefore, the development of ADCs for therapeutic purposes has been more challenging.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has a structure of the general formula (Pc-L-Y-D):

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -O-CR ¹ R ² -(CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O);

R ^a and R ^b are the same or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclyl;

or R ^a and R ^b , together with the carbon atoms bound thereto, form cycloalkyl or heterocyclyl;

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

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;

or, R ¹ and R ² , together with the carbon atoms bound thereto, form cycloalkyl or heterocyclyl;

or, R ^a and R ² , together with the carbon atoms attached thereto, form cycloalkyl or heterocyclyl;

m is an integer between 0 and 4;

n is a decimal or integer number between 1 and 10;

L is a linker moiety;

Pc is an antibody or its antigen-binding fragment;

wherein said composition has a pH from about 4.5 to about 6.0, preferably a pH from about 4.8 to about 5.3, and more preferably a pH from about 5.0 to about 5.1.

In an alternative embodiment, the buffer in the pharmaceutical composition has a pH of about 4.5 to about 6.0; non-limiting examples include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0; about 4.8 to about 5.3 is preferred, and about 5.0 to about 5.1 is more preferred. In some embodiments, the pharmaceutical composition has a pH of 4.5 to 5.2, preferably a pH of 4.8 to 5.2, and more preferably a pH of 5.0 to 5.1. In some embodiments, the pharmaceutical composition has a pH of 5.0.

In an alternative embodiment, the pharmaceutical composition further comprises a surfactant that may be selected from the group consisting of polysorbate, polysorbate 20, polysorbate 80, poloxamer, triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octylglycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauramidopropyl betaine, cocaramidopropyl betaine, linolein amidopropyl betaine, myristyl amidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristyl amidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, ethylene propylene glycol copolymer, etc. The surfactant is preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In an alternative embodiment, the surfactant in the pharmaceutical composition is present at a concentration of about 0.01 mg/mL to about 1.0 mg/mL. In an alternative embodiment, the surfactant in the pharmaceutical composition is present at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, preferably about 0.1 mg/mL to about 0.3 mg/mL, about 0.2 mg/mL to about 0.6 mg/mL, about 0.2 mg/mL to about 0.5 mg/mL, or about 0.2 mg/mL to about 0.3 mg/mL, and more preferably about 0.2 mg/mL; non-limiting examples include 0.1 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45 mg/mL, 0.5 mg/mL, and 0.6 mg/mL.

In an alternative embodiment, the above pharmaceutical composition further comprises a saccharide. The "saccharide" of the present invention includes the general composition ( _CH2O ) _n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The saccharide may be selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc. The saccharide is preferably a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.

In an alternative embodiment, the saccharide in the above pharmaceutical compositions is present at a concentration of about 60 mg/mL to about 90 mg/mL; non-limiting examples include 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, and 90 mg/mL; preferably 80 mg/mL. In some embodiments, the saccharide is present at a concentration of 70 mg/mL to 90 mg/mL.

In an alternative embodiment, the antibody-drug conjugate in the pharmaceutical composition has a concentration of from about 1 mg/mL to about 100 mg/mL; non-limiting examples include 1 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, and 100 mg/ml; preferably from about 10 mg/ml to about 30 mg/ml, and more preferably from about 20 mg/ml to about 22 mg/ml. In particular, non-limiting examples include 20.1 mg/ml, 20.2 mg/ml, 20.3 mg/ml, 20.4 mg/ml, 20.5 mg/ml, 20.6 mg/ml, 20.7 mg/ml, 20.8 mg/ml, 20.81 mg/ml, 20.82 mg/ml, 20.83 mg/ml, 20.84 mg/ml, 20.85 mg/ml, 20.86 mg/ml, 20.87 mg/ml, 20.88 mg/ml, 20.89 mg/ml, 20.9 mg/ml, 20.91 mg/ml, 20.92 mg/ml, 20.93 mg/ml, 20.94 mg/mL, 20.95 mg/mL, 20.96 mg/mL, 20.97 mg/mL, 20.98 mg/mL, 20.99 mg/mL, and 21 mg/mL. In an alternative embodiment, the antibody drug conjugate in the pharmaceutical composition is present at a concentration of about 10 mg/mL to about 30 mg/mL, preferably about 20 mg/mL, based on the naked antibody (i.e., the antibody portion of the ADC).

In an alternative embodiment, the buffer in the above-mentioned pharmaceutical composition is selected from the group consisting of a histidine salt buffer, a succinate buffer and a citrate buffer, preferably a succinate buffer and more preferably a succinic acid-sodium succinate buffer.

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

In an alternative embodiment, the drug content (n) may be from 3 to 8, from 4 to 8, from 5 to 7, more preferably from 5.3 to 6.1, 5.7 cytotoxic drugs binding to each antibody or antigen-binding fragment thereof (Pc), n being a decimal or integer.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) an antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; wherein the pharmaceutical composition has a pH of about 4.8 to about 5.3.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) an antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; wherein the pharmaceutical composition has a pH of 4.8-5.2.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) an antibody drug conjugate at a concentration of about 20 mg/mL to about 22 mg/mL, (b) polysorbate 80 at a concentration of about 0.2 mg/mL, (c) sucrose at a concentration of 80 mg/mL, and (d) a succinate buffer at a concentration of 10 mM; wherein the pharmaceutical composition has a pH of about 5.0 to about 5.1. In some embodiments, the pharmaceutical composition has a pH of 5.0-5.1.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- represents -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R ^a and R ^b are the same or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl;

R ¹ is C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

m represents 0 or 1.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- represents -O-(CH ₂ ) _m -CR ¹ R ² -C(O)-;

R ¹ is C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

m represents 0 or 1.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- represents -O-(CH ₂ ) _m -CR ¹ R ² -C(O)-;

R ¹ is C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

R ² is hydrogen;

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

m represents 0 or 1.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- represents -O-(CH ₂ ) _m -CR ¹ R ² -C(O)-;

R ¹ is C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

R ² is hydrogen;

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

m represents 0.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- is selected from the group consisting of:

, , , , , And .

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

The O-terminus of Y is attached to the linker fragment L.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate,

-Y- is selected from the group consisting of:

, , , , , And .

In an alternative embodiment, the above-mentioned antibody-drug conjugate has a structure of the general formula (Pc-LD ₁ ):

Where:

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

m represents 0 or 1;

n can be an integer or a decimal number from 1 to 10;

Pc is an antibody or an antigen-binding fragment thereof; and L is a linker moiety.

In an alternative embodiment of the invention, in the above-mentioned antibody-drug conjugate, n may be an integer or decimal number from 2 to 8, preferably an integer or decimal number from 3 to 8.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the linker moiety -L- is -L ¹ -L ² -L ³ -L ⁴ -, where:

L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-WC(O)-, -CH ₂ -C(O)-NR ³ -WC(O)- and -C(O)-WC(O)-, wherein W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl cycloalkyl and linear heteroalkyl of 1 to 8 atoms, and the heteroalkyl contains 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein each of the C _1-8 alkyl, cycloalkyl and linear heteroalkyl is independently further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and 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 ² is preferably a chemical bond;

L ³ is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ )t-, -C(O)NR ⁵ , -C(O)NR ⁵ (CH ₂ )t- and a chemical bond, where t is an integer from 1 to 6; L ⁴ is preferably -NR ⁵ (CR ⁶ R ⁷ )t-;

R ³ , R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In an alternative embodiment, in the above antibody drug conjugate, the linker moiety L ¹ is selected from the group consisting of -(succinimidin-3-yl-N)-(CH ₂ )s ¹ -C(O)-, -(succinimidin-3-yl-N)-CH ₂ -cyclohexyl-C(O)-, -(succinimidin-3-yl-N)-(CH ₂ CH ₂ O)s ² -CH ₂ CH ₂ -C(O)-, -CH ₂ -C(O)-NR ³ -(CH ₂ )s ³ -C(O)- and -C(O)-(CH ₂ )s ⁴ C(O)-, wherein s ¹ is an integer from 2 to 8, s ² is an integer from 1 to 3, s ³ is an integer from 1 to 8, and s ⁴ is an integer from 1 to 8; s ¹ is preferably 5.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the linker moiety L ² is -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ C(O)- or a chemical bond, and p ¹ is an integer from 6 to 12.

In an alternative embodiment, in the above antibody-drug conjugate, L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is hydrogen or alkyl, R ⁶ and R ⁷ are the same or different and each independently is hydrogen or alkyl, and t is 1 or 2, preferably 2; L ⁴ is preferably -NR ⁵ CR ⁶ R ⁷ -, more preferably -NHCH ₂ -.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the linker moiety -L- is -L ¹ -L ² -L ³ -L ⁴ -, where:

L ¹ is and s ¹ is an integer from 2 to 8;

L ² is a chemical bond;

L ³ is a tetrapeptide residue;

L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is hydrogen or alkyl, R ⁶ and R ⁷ are the same or different and each independently is hydrogen or alkyl, and t is 1 or 2.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the linker moiety -L- is -L ¹ -L ² -L ³ -L ⁴ -, where:

L ¹ is -(succinimidin-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

L ³ is a tetrapeptide residue;

L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is hydrogen or alkyl, R ⁶ and R ⁷ are the same or different and each independently is hydrogen or alkyl, and t is 1 or 2.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the peptide residue L ³ is an amino acid residue formed from one, two or more amino acids selected from the group consisting of phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (N), preferably an amino acid residue formed from one, two or more amino acids selected from the group consisting of phenylalanine and glycine, more preferably a tetrapeptide residue and most preferably a GGFG (glycine-glycine-phenylalanine-glycine) tetrapeptide residue.

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, the linker moiety -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein the L ¹ -end is linked to the antibody or antigen-binding fragment thereof and the L ⁴ -end is linked to Y.

In an alternative embodiment, in the above antibody-drug conjugate, -L-Y- is:

L ¹ is -(succinimidin-3-yl-N)-(CH ₂ )s ¹ -C(O)- or -(succinimidin-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ C(O)- or a chemical bond, and p ¹ is an integer from 6 to 12; R ⁴ is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

L ³ is a residue of the tetrapeptide GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl;

s ¹ is an integer from 2 to 8, preferably 5;

m is an integer between 0 and 4.

In an alternative embodiment, in the above antibody-drug conjugate, -L-Y- is:

preferably:

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-; R ⁴ is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

L ³ is a residue of the tetrapeptide GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl;

m is an integer between 0 and 4.

In an alternative embodiment, in the above antibody-drug conjugate, -L-Y- is:

L ² is a chemical bond;

L ³ is a residue of the tetrapeptide GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl;

s ¹ is an integer from 2 to 8, preferably 5;

m is an integer between 0 and 4.

In an alternative embodiment, in the above antibody-drug conjugate, -L-Y- is:

Where:

L ¹ is -(succinimidin-3-yl-N)-(CH ₂ )s ¹ -C(O)- or -(succinimidin-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) p ¹ CH ₂ C(O)- or a chemical bond, and p ¹ is an integer from 1 to 20; R ⁴ is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

L ³ is a residue of the tetrapeptide GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

R ⁵ , R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl;

s ¹ is an integer between 2 and 8;

m is an integer between 0 and 4.

In an alternative embodiment, in the above antibody-drug conjugate, -L-Y- is:

,

Where:

L ² is a chemical bond;

L ³ is a residue of the tetrapeptide GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-7 cycloalkylalkyl or C _3-7 cycloalkyl;

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

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

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl;

s ¹ is an integer between 2 and 8;

m is an integer between 0 and 4.

In an alternative embodiment, the above-mentioned antibody-drug conjugate has a structure of 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 containing from 1 to 8 atoms, and the heteroalkyl contains from 1 to 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 further optionally 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; R ⁴ is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

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 thereto, 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 represents 0 or 1;

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

Pc is an antibody or its antigen-binding fragment.

In an alternative embodiment, the above-mentioned antibody-drug conjugate has a structure of the general formula (Pc-L _b -YD):

Where:

s ¹ is an integer between 2 and 8;

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

In an alternative embodiment, in the above-mentioned antibody-drug conjugate, -L-Y- includes, but is not limited to:

In an alternative embodiment, the above-mentioned antibody-drug conjugate has the following structure:

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

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

,

Where:

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

Pc is an antibody or its antigen-binding fragment.

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

In an alternative embodiment, the Pc is selected from the group consisting of an anti-HER2 (epidermal growth factor receptor 2) antibody, an anti-EGFR (epidermal growth factor receptor 1) antibody, an anti-B7-H3 (B7 homolog protein 3) antibody, an anti-c-Met (hepatocyte growth factor receptor) antibody, an anti-HER3 (epidermal growth factor receptor 3) antibody (ErbB3), an anti-HER4 (epidermal growth factor receptor 4) antibody (ErbB4), 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 (Crypto), an anti-EphA2 antibody, an anti-G250 antibody, an anti- MUCl, anti-Lewis Y antibodies, anti-VEGFR (vascular endothelial growth factor) antibodies, anti-GPNMB (glycoprotein B of non-metastatic melanoma) antibodies, anti-integrin antibodies, anti-PSMA (prostate-specific membrane antigen) antibodies, anti-tenascin-C antibodies, anti-SLC44A4 (choline transporter-like protein 4) antibodies, anti-mesothelin antibodies or their antigen-binding fragments.

In an alternative embodiment, the antibody or antigen-binding fragment thereof in the antibody-drug conjugate is selected from the group consisting of trastuzumab, pertuzumab, nimotuzumab, enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96 and glematumamab or antigen-binding fragments thereof.

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

where n is an integer or decimal number from 0 to 10, not equal to zero, preferably an integer or decimal number from 1 to 10, more preferably an integer or decimal number from 2 to 8, and most preferably an integer or decimal number from 3 to 8.

In an alternative embodiment, the antibody-drug conjugate in the pharmaceutical composition has the following structure:

,

where n is a decimal or integer number from 3 to 8.

The present invention provides a pharmaceutical composition comprising: (a) an antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; wherein the composition has a pH of 4.8-5.2,

where the antibody-drug conjugate has the following structure:

,

where n is a decimal or integer number from 3 to 8.

The present invention provides a pharmaceutical composition comprising (a) an antibody-drug conjugate at a concentration of about 20 mg/mL to about 22 mg/mL, (b) polysorbate 80 at a concentration of about 0.2 mg/mL, (c) sucrose at a concentration of about 80 mg/mL, and (d) a succinate buffer at a concentration of about 10 mM; wherein the pharmaceutical composition has a pH of 5.0-5.1,

where the antibody-drug conjugate has the following structure:

,

where n is a decimal or integer number from 3 to 8.

The present description also provides a lyophilized preparation comprising a conjugate of an antibody and a drug, wherein the preparation can be reconstituted to form the pharmaceutical composition described above.

The present invention also provides a method for producing a lyophilized preparation containing an antibody-drug conjugate, comprising a step of lyophilizing the pharmaceutical composition described above.

In an alternative embodiment, the lyophilization in the method for producing a lyophilized preparation comprising an antibody-drug conjugate comprises the steps of pre-freezing, primary drying and secondary drying. Lyophilization is carried out by freezing the preparation and then sublimating the water at a temperature suitable for primary drying. Under these conditions, the product is at a temperature below the eutectic point or the destruction temperature of the preparation. Typically, the temperature for primary drying is from about -30 °C to 25 °C (provided that the product remains frozen during primary drying). The preparation, the size and type of container (e.g., a glass vial) containing the sample and the volume of liquid determine the time required for drying, which can be from several hours to several days (e.g., 40-60 hours). Secondary drying can be carried out at a temperature of about 0-40 °C, depending primarily on the type and size of the container and the type of protein used. The time required for secondary drying is determined by the desired residual water content of the product and typically takes at least about 5 hours. Typically, the water content of a preparation lyophilized at low pressure is less than about 5%, preferably less than about 3%. The pressure may be the same as that of the primary drying stage; preferably, the secondary drying pressure is lower than the primary drying pressure. Lyophilization conditions may vary depending on the composition and size of the vial.

In an alternative embodiment of the present invention, 5 ml of the stock solution of the pharmaceutical composition are lyophilized, and the lyophilization program is as follows: for preliminary freezing, the temperature is -5 °C or -45 °C; for primary drying, the temperature is -20 °C, and the degree of vacuum is 10 Pa; for secondary drying, the temperature is 25 °C, and the degree of vacuum is 1 Pa.

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

The present invention also provides a lyophilized preparation comprising an antibody-drug conjugate obtained by lyophilizing a pharmaceutical composition comprising an anti-HER2 antibody-drug conjugate described above.

The present invention also provides a reconstituted solution containing an antibody-drug conjugate obtained by reconstituting the lyophilized preparation described above.

The present invention also provides a method for producing the reconstituted solution described above, comprising the step of reconstituting the above-mentioned lyophilized preparation with a solution selected from the group consisting of, but not limited to, water for injection, physiological saline or glucose solution.

In an alternative embodiment, the reconstituted solution comprises the following components:

(a) an antibody-drug conjugate at a concentration of from about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at a concentration of from about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at a concentration of from about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of from about 5 mM to about 20 mM; wherein the reconstituted solution has a pH of 4.8-5.2.

In an alternative embodiment, the reconstituted solution comprises the following components:

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

The present invention also provides an article comprising a container comprising a pharmaceutical composition, a lyophilized preparation or a reconstituted solution as described above. In some embodiments, the container is a tubular injection vial made of neutral borosilicate glass.

The invention also proposes the use of the above-mentioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or article for obtaining a medicinal product for the treatment or prevention of tumors.

The present invention also provides a method of treating a disease, comprising providing the above-mentioned pharmaceutical composition or lyophilized preparation or reconstituted solution or article.

The present invention also provides the above-mentioned pharmaceutical composition or lyophilized preparation or reconstituted solution or article as a medicinal product, preferably for treating or preventing a tumor disease.

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

In an alternative embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland carcinoma, esophageal cancer, melanoma, neuroglioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

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

The present invention provides a pharmaceutical composition that facilitates production and administration and is stable in properties. In particular, the pharmaceutical composition described in the present description comprises a conjugate of an antibody and a drug and a buffer.

Terms

To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention pertains.

Application PCT/CN2019/107873 (WO2020/063676) is incorporated herein by reference in its entirety.

"Antibody drug conjugate (ADC)" was prepared by combining an antibody or an antibody fragment with a cytotoxin with biological activity or a small molecule drug with cell killing activity by a stable chemical linker compound, in which case the binding specificity of the antibody to tumor cell-specific antigens or highly expressed antigens and the high efficiency of the cytotoxin are fully realized, and toxic side effects on normal cells are avoided. The antibody drug conjugate can accurately bind to tumor cells and have a reduced effect on normal cells compared with conventional chemotherapeutic drugs used previously.

"Buffer" refers to a buffer that resists changes in pH through the action of its acid-base conjugate components. Examples of buffers that control 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 hydrochloride buffer, histidine acetate buffer, histidine phosphate buffer, histidine sulfate buffer, etc., and histidine acetate buffer is preferred. Histidine acetate buffer was prepared from histidine and acetic acid, and histidine hydrochloride buffer was prepared from histidine and hydrochloric acid.

"Citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. Preferably, the citrate buffer is citric acid-sodium citrate.

"Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, etc. A preferred succinate buffer is succinic acid-sodium succinate. As an example, a succinic acid-sodium succinate buffer can be prepared from succinic acid and sodium hydroxide or from succinic acid and sodium succinate.

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

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

"Pharmaceutical composition" refers to a mixture comprising one or more antibody-drug conjugates described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, with other components being, for example, physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active antibody ingredient and to facilitate administration into the body, thereby facilitating absorption of the active ingredient, thereby exhibiting its biological activity.

The terms "pharmaceutical composition" and "preparation" as used herein are not mutually exclusive.

The pharmaceutical composition described in the present invention is in the form of a solution, and unless otherwise indicated, the solvent is water.

"Lyophilized preparation" refers to a preparation or pharmaceutical composition obtained by vacuum lyophilization of a pharmaceutical composition or preparation in liquid or dissolved form.

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 the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation according to practice 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 provided in this application and the claims, unless otherwise indicated, the meaning of "about" or "substantially comprising" should be understood to mean within the acceptable range of error for that particular value.

The pharmaceutical composition of the present invention can achieve a stable effect: a pharmaceutical composition in which the antibody-drug conjugate substantially maintains its physical and/or chemical stability and/or biological activity after storage; preferably, the pharmaceutical composition substantially maintains its physical and chemical stability, as well as its biological activity after storage. The storage period was generally selected based on a predetermined shelf life of the pharmaceutical composition. Currently, there are many analytical techniques available for measuring protein stability, and the stability after storage for a selected period of time at a selected temperature can be measured.

A stable preparation is a preparation that does not show significant changes under the following conditions: storage at refrigerated temperature (2-8 °C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, stable liquid preparations include liquid preparations that exhibit desirable properties after storage at temperatures including 25 °C for periods including 1 month, 3 months, and 6 months. Typical examples of stability are as follows: generally no more than about 10%, preferably no more than about 5% of the antibody monomers aggregate or decompose, as measured by size-exclusion SEC-HPLC (size-exclusion high-performance liquid chromatography). The preparation is a pale yellow, almost colorless and transparent liquid, or colorless, or transparent, or slightly opalescent, when visually analyzed. The concentration, pH and osmotic pressure of the preparation change by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, decreases are observed. Typically, no more than about 10%, preferably no more than about 5%, aggregation is formed.

An antibody-drug conjugate "retains its physical stability" in a pharmaceutical preparation if it does not exhibit significant increases in aggregation, precipitation, and/or denaturation as determined visually by color and/or clarity, or as measured by ultraviolet light scattering (UV), size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and FTIR (Fourier transform infrared spectroscopy) spectroscopy (which determines the secondary structure of the protein).

An antibody-drug conjugate "retains its chemical stability" in a pharmaceutical preparation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of proteins include hydrolysis or clipping (assessed by techniques such as size exclusion chromatography and CE-SDS (sodium dodecyl sulfate capillary electrophoresis)), oxidation (assessed by techniques such as peptide mapping coupled to mass spectroscopy or MALDI/TOF/MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), deamidation (assessed by techniques such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (assessed by isoaspartic acid measurement, peptide mapping, etc.).

An antibody-drug conjugate "retains its biological activity" in a pharmaceutical preparation if the biological activity of the antibody-drug conjugate at a given time is within the specified range of biological activity exhibited during the preparation of the pharmaceutical preparation.

The three-letter and one-letter amino acid codes used in this document are described in J. Biol. Chem., 243, p. 3558 (1968).

"An antibody" as described herein refers to an immunoglobulin, and an intact antibody is a tetrapeptide chain structure formed by the linkage between two identical heavy chains and two identical light chains via interchain disulfide bonds. The constant regions of an immunoglobulin heavy chain differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be divided into five classes, otherwise called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with their respective heavy chains being the μ chain, δ chain, γ chain, α chain and ε chain, respectively. An Ig of one class can be divided into different subclasses depending on differences in the amino acid composition of the hinge regions and the number and arrangement 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 based on 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 a specific antibody against cell surface antigens on target cells, non-limiting examples of antibodies being one or more 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-CEA ... anti-PSMA, anti-tenascin-C antibody, anti-SLC44A4 antibody and anti-mesothelin antibody, preferably trastuzumab (under the trade name Herceptin), pertuzumab (also known as 2C4, under the trade name Perjeta), nimotuzumab (under the trade name Taixinsheng), enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96 and glematumamab.

In the heavy and light chains of antibodies, the sequences of about 110 amino acids near the N-terminus vary considerably and are thus called variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are thus called constant regions. The variable region includes 3 hypervariable regions (HVRs) and 4 framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are thus 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 three CDR regions of the light chain are designated LCDR1, LCDR2 and LCDR3, the three CDR regions of the heavy chain are designated HCDR1, HCDR2 and HCDR3. The amino acid residues of the CDR regions LCVR and HCVR of the antibodies or antigen-binding fragments described herein correspond to the known Kabat numbering scheme (LCDR 1-3, HCDR 1-3) in number and position.

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 a mouse in accordance with the knowledge and experience in the art. During production, the subject is administered an antigen, and then hybridomas expressing antibodies with the desired sequences 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 induced by the mouse antibody. The chimeric antibody is created by first creating a hybridoma secreting a mouse-specific monoclonal antibody, then cloning the variable region gene from mouse hybridoma cells, cloning the constant region gene of a human antibody as needed, fusing the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing the chimeric antibody molecules in a eukaryotic production system or a prokaryotic production system.

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into the variable region framework of a human antibody, i.e., another type of antibody framework sequence from a human species. Such an antibody can overcome the strong heterogeneous response induced by the chimeric antibody due to the transfer of a large number of mouse 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 species sequence database "VBase" and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the FR (framework region) sequence in the variable region of a human antibody may be subjected to minimal reversible or reverse mutation to maintain activity. The humanized antibody of the present invention also includes humanized antibodies that have further undergone CDR affinity maturation using phage display.

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

"Antigen-binding fragment of an antibody" as described herein may refer to a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, or an scFv fragment, an Fv fragment that binds to an antigen that has antigen-binding activity. An Fv fragment comprises the variable region of the heavy chain and the variable region of the light chain of an antibody, but without the constant region, and has the smallest antibody fragment of all the antigen-binding sites. Typically, an Fv antibody also comprises a polypeptide linker between the VH (heavy chain variable domain) and VL (light chain variable domain) 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, means that cells expressing an Fc receptor directly kill target cells coated with an antibody by recognizing the Fc segment of the antibody. The ADCC effector function of an antibody can be reduced or eliminated by modifying 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 L234A/E235A of IgG4.

"Mutations" in the mutant sequences described herein include, but are not limited to, a "backmutation," a "conservative modification," or a "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 conformation and rigidity of the backbone), such that the changes can often be made without altering the biological activity of the protein. Those skilled in the art will recognize that, in general, the replacement of a single amino acid in a non-essential region 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., p. 224, (4th edition)). Furthermore, substitution of amino acids with similar structure or function is unlikely to impair biological activity.

The "mutant sequence" described herein refers to a nucleotide sequence and/or amino acid sequence having varying degrees of percent sequence 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 identity of the sequence described herein may be at least 85%, 90% or 95%, and preferably at least 95%. Non-limiting examples include: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. Sequence comparison and determination of percent identity between two sequences can be performed using default settings or the BLASTN/BLASTP algorithm available on the National Center for Biotechnology Institute website.

The term "linker unit" or "linker fragment" refers to a fragment of a chemical structure or bond 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 it is linked to another linker. In preferred embodiments of the present invention, they are designated as L and L ¹ -L ⁴ , where the L ¹ end is linked to the antibody and the L ⁴ end is linked to the Y structural unit and then to the compound or toxin.

The linker may comprise stretcher units, spacer units, and amino acid units, and may be synthesized using methods known in the art, such as those described in US2005-0238649A1. The linker may 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 may 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 in the present invention can be produced and purified by conventional methods. For example, cDNA (complementary DNA) 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 (Chinese hamster ovary cells). As more recommended in the prior art, mammalian expression systems can lead to glycosylation of antibodies, in particular, at the highly conserved N-terminal site of the Fc region. Positive clones were cultured in serum-free medium of an antibody production bioreactor. The culture with secreted antibody can be purified and collected using conventional techniques. For example, purification was carried out on an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound fractions were washed out. The bound antibody was eluted by a pH gradient method and the antibody fragments were 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, e.g. 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, various branched isomers, etc. More preferably, the lower alkyl contains from 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl 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 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 "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 moieties derived from a parent alkane by the removal of 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, 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 the group -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, 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 (wherein m is an integer from 0 to 2), except for the cyclic portion -OO-, -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 share one atom (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, spiroheterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. According to the number of spiroatoms shared between the rings, spiroheterocyclyl may be monospiroheterocyclyl, bispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and bispiroheterocyclyl, and 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 a system wherein one or more rings may contain one or more double bonds but none 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 fused heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. According to 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 the fused heterocyclyl include:

And .

The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two carbon atoms that are not directly attached 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. According to the number of rings formed, the bridged heterocyclyl may 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 connected to the parent structure is a heterocyclyl. Non-limiting examples of a heterocyclyl ring include:

, 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., rings sharing 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, wherein the ring attached to the parent structure is an aryl ring. Non-limiting examples of an aryl ring include:

And .

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, such as 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 connected to the parent structure is a heteroaryl ring. Non-limiting examples of a heteroaryl ring include:

And .

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 the reaction is carried out elsewhere in the molecule. Non-limiting examples of amino protecting groups 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 halogens, 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 "optional" or "optionally" means that the event or circumstance subsequently described may, but need not, 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 an antibody heavy chain variable region of a particular sequence may, but need not, be present.

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

The term "drug loading" refers to the average number of cytotoxic drugs loaded onto each antibody or antigen-binding fragment thereof in the antibody-drug conjugate molecules, and it can also be expressed as a ratio of the number of drugs to the number of antibodies. The drug loading can be from 0 to 12, preferably from 1 to 10, more preferably from 3 to 8 and most preferably from 5.3 to 6.1 cytotoxic drugs bound to each antibody or antigen-binding fragment thereof (Pc). In embodiments of the present invention, the drug loading is represented in n, which can also be referred to as the DAR (drug-to-antibody ratio) value, and exemplary values can be average values of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drugs per ADC molecule in binding reactions can be characterized by conventional methods such as UV-Vis spectroscopy, hydrophobic interaction chromatography with mass spectrometry (HIC), ELISA (enzyme-linked immunosorbent assay), and HPLC.

Cytotoxic drug loading may be controlled by the following non-limiting methods, including:

(1) control of the molar ratio of linker reagent to monoclonal antibody,

(2) control of reaction time and temperature, and

(3) selection of different reagents.

For generally accepted pharmaceutical compositions, reference is made to the Chinese Pharmacopoeia.

The term "carrier" for a drug according to the present invention refers to a system that can change the way a drug enters the human body and the distribution of the drug in the human body, control the release rate of the drug, and deliver the drug to the target organ. The release and targeting system based on the drug carrier can reduce the degradation and loss of the drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structures to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals, and vesicles, as preferred examples. The aggregates have the ability to encapsulate drug molecules and have good membrane permeability, and therefore can be used as excellent drug carriers.

The terms "treat" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with animals, humans, subjects, cells, tissues, organs, or biological fluids. The terms "treat" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, investigational, and experimental methods. Treating cells includes contacting a reagent with cells and contacting a reagent with a fluid. The terms "treat" and "treatment" also refer to treating, for example, cells with reagents, diagnostics, binding compositions, or another cell in vitro and ex vivo. "Treatment" when applied to humans, veterinary, or investigational subjects refers to therapeutic treatment, preventive or prophylactic measures, and investigational and diagnostic uses.

"Treat" or "treatment" refers to the administration of a therapeutic agent, such as a composition comprising any of the conjugate compounds of the present invention, either internally or externally to a patient with one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population treated, to induce regression of such symptoms, or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors, such as the state of the disease, the age and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Alleviation of a symptom of a disease 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 a symptom. Although embodiments of the present invention (e.g., treatment methods or products) may not be effective in alleviating the symptoms of each disease of interest, they should reduce 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 and Whitney U-test, the Kruskal-Wallis test (H-test), the Jonker-Terpstra test, and the Wilcoxon test.

"An effective amount" includes an amount sufficient to alleviate or prevent the occurrence of 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 and dosage of administration, and the severity of adverse effects. An effective amount may be the maximum dose or mode of administration to avoid significant adverse effects or toxic effects.

"Exchange" refers to the exchange of the solvent system that solubilizes the antibody protein. For example, a high salt or hypertonic solvent system containing the antibody protein is exchanged by physical operations with a buffer system of a stable preparation so that the antibody protein is present in a stable preparation. Physical operations include, but are not limited to, ultrafiltration, dialysis, or recovery from centrifugation.

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 the plasma stability test for ADC-18 of the present invention.

Fig. 1C shows the results of the plasma stability test for ADC-20 of the present invention.

Fig. 2 shows the efficacy evaluation of ADC-21 and ADC-24 of the present invention in JIMT-1 tumor-bearing mice.

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

Fig. 4 shows the results of the plasma stability test for ADC-25 of the present invention.

Fig. 5 shows the efficacy of the ADC of the present invention against human brain astrocytoma tumor U87MG xenografted into nude mice.

Fig. 6 shows the efficacy of the ADC of the present invention against Detroit 562 human metastatic pharyngeal cancer tumor cells with pleural effusion xenografted into nude mice.

Fig. 7 shows the efficacy of the ADC of the present invention against human U87MG glioblastoma brain tumor xenografted into nude mice.

Figure 8 shows the fitted trend plots for the formula screening experiment where PS80 is in units of 10 ^-4 g/mL and the ADC-32 protein concentration (based on naked antibody) is in units of mg/mL.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described below with reference to examples, which, however, are not intended to limit the scope of the present invention. The experimental methods in the examples of the present invention, in which specific conditions are not indicated, are generally carried out under conventional conditions, for example, with reference to Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual from Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer of the raw materials or goods. Reagents without indication of a specific origin are commercially available conventional reagents.

I. Antibody-drug conjugates

The structures of the compounds were identified using nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ) and deuterated methanol ( _CD3OD ) as solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in units of 10 ^-6 ( ^ppm ).

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

UPLC (ultra-performance liquid chromatography) analysis was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

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

UV-HPLC (high performance liquid chromatography with UV detector) analysis was performed using a Thermo nanodrop2000 UV spectrophotometer.

The degree of proliferation inhibition and IC ₅₀ (half-maximal inhibitory concentration) values were measured using a PHERA starFS microplate reader (BMG, Germany).

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

Yantai Huanghai 200-300 mesh silica gel was commonly used as a carrier in column chromatography.

The known starting materials of the present invention can be synthesized using or according to 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, all reactions were carried out under argon or nitrogen atmosphere unless otherwise stated.

Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of argon or nitrogen.

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

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

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

A CEM Discover-S 908860 microwave reactor 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 to 30 °C.

Preparation of PBS (phosphate buffered saline) buffer at pH 6.5 in _the examples: 8.5 g _KH2PO4 , 8.56 g _K2HPO4.3H2O , _5.85 g NaCl and 1.5 g EDTA (ethylenediaminetetraacetic acid) _were added to the flask and the volume was adjusted to 2 L. All additions were dissolved by ultrasound and the solution was mixed well by shaking to obtain the required buffer.

The elution system for column chromatography and the developing solvent system for thin layer chromatography used for the purification of the compound included: A: dichloromethanol and isopropanol system, B: dichloromethanol and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or a small amount of triethylamine and an acidic or basic reagent was added.

Some of the compounds of the present invention were characterized by Q-TOF LC/MS (liquid chromatography with mass spectrometry) analysis. Q-TOF LC/MS analysis was performed on an Agilent 6530 accurate mass quadrupole time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm chromatography column).

Example 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 as described in patent application "EP0737686A1") was added 1 ml of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by a 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 addition, the reaction mixture 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 using developing solvent system B to give the title product 1 (1.6 mg, yield 82.1%).

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

^1H 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 compound 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The system was purged with argon three times, and 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 mixture was stirred until it became clear. To the reaction mixture were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared as described 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 the addition, the reaction mixture was stirred at 0-5 °C for 1 h. The ice water bath was removed and the reaction mixture was warmed 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 products (2-A: 1.5 mg, 2-B: 1.5 mg).

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

Compound 2-B with a single configuration (with a shorter retention time):

UPLC analysis: retention time: 1.06 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 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 8.37 (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 with single 2-A configuration (with longer retention time):

UPLC analysis: retention time: 1.10 min; purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 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 compound 1b (5.0 mg, 9.41 μmol) were added 2 ml of ethanol and 0.4 ml of N,N-dimethylformamide, and 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 mixture was stirred until it became clear. To the reaction mixture were added 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, purchased from Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially. After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture 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 products 1.5 mg, 1.5 mg).

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

Single configuration compound (with shorter retention time):

UPLC analysis: retention time: 1.11 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 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).

Single configuration compound (with longer retention time):

UPLC analysis: retention time: 1.19 min; purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 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 compound 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 and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared as described in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) sequentially. After addition, the reaction mixture 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 using developing solvent system B to give the title product 4 (2.5 mg, yield 80.9%).

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

¹ H NMR (400 MHz, 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 compound 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 and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared as described in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) sequentially. After addition, the reaction mixture 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 using developing solvent system B to give the title product 5 (1.0 mg, yield 50%).

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

^1H NMR (400 MHz, 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 compound 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 and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, prepared as described in "Journal of the American Chemical Society, 2014, vol.136, #22, p. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After addition, the reaction mixture 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 using developing solvent system B to give the title product 6 (2.1 mg, yield 67.9%).

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

¹ H NMR (400 MHz, DMSO-d ₆ ): 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-hydroxycyclobutane-1-carboxamide 7

To compound 1b (3.0 mg, 5.64 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and 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 mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, purchased from PharmaBlock), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol). After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture 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%).

MS 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-pentaazeicosyl)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)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol; prepared as described in patent application "US2005/20645") and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methyl acetate 8b (100 mg, 0.27 mmol; prepared as described in patent application "CN105829346A") were added to the reaction flask and 5 mL of tetrahydrofuran was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of potassium tert-butoxide (61 mg, 0.54 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by the addition of 20 ml of ice water and extraction 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 mixture was stirred at room temperature for 1 h. 20 ml of water was added, followed by extraction 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%).

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

Stage 2

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

Compound 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 palladium on carbon (25 mg, 10% loading) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture 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,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

Compound 1b (7 mg, 0.013 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine, a solution of compound 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 mixture was stirred in an ice bath for 35 min. 10 ml of water was added, followed by extraction with ethyl acetate (5 ml×3). The organic phases were combined, 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%).

MS 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

Compound 8e (4 mg, 4.84 μmol) was dissolved in 0.2 ml of dichloromethane and 0.1 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the 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-pentaazeicosyl)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 system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylamino)acetylamino)-3-phenylpropionic acid 8 g (2.7 mg, 5.80 μmol, prepared as described in patent application "EP2907824") 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 mixture was stirred in an ice bath for 30 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature, stirred for 15 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 8 (2 mg, yield 39.0%).

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

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

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

Compound 2a (1.3 g, 11.2 mmol; prepared as described in patent application "WO2013/106717") was dissolved in 50 mL of acetonitrile and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 h and 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 g, 86.9% yield).

Stage 2

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

Compound 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to the reaction flask and 4 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (109 mg, 0.98 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 40 min followed by the addition of 10 mL of ice water and extraction 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 thereto. The reaction mixture was stirred at room temperature for 2 h. 20 ml of water was added, followed by extraction 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 C developing solvent system 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

Compound 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 on carbon (12 mg, 10% loading, dry substance) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture 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 used directly in the next step without purification.

MS 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

Compound 1b (10 mg, 18.8 μmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of crude triethylamine 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 mixture was stirred in an ice bath for 40 min. 10 ml of water was added, followed by extraction 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%).

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

Stage 5

2-((2-aminoacetylamino)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioc co-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

Compound 9d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left to stand. Then, the supernatant was removed and a solid was left. The solid residue was concentrated under reduced pressure and dried using an oil pump to give the crude title product 9e (17 mg), which was directly used in the next step without purification.

MS 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 system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of 8 g (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 mixture was stirred in an ice bath for 10 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 1 h to give compound 9. The reaction mixture 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 give the title products (9-A: 2.4 mg, 9-B: 1.7 mg).

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

Compound 9-A with a single configuration (with a shorter retention time):

UPLC analysis: retention time: 1.14 min; purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 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), 2.04-2.15 (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 with single configuration 9-B (with longer retention time):

UPLC analysis: retention time: 1.16 min; purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 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-B

Stage 1

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

Compound 3a (1.80 g, 12.5 mmol) was dissolved in 100 mL of acetonitrile, and potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol), and tetrabutylammonium iodide (231 mg, 0.63 mmol) were added successively. The reaction mixture was heated to 60 °C, 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 system C developing solvent to give the title product 10a (980 mg, yield 33.5%).

^1H 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

Compound 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to the reaction flask and 3 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (38 mg, 0.34 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 20 min followed by the addition of 10 mL of ice water and extraction with ethyl acetate (20 mL×2) and chloroform (10 mL×5). The organic phases were combined and concentrated. The obtained residue was dissolved in 2 ml of dioxane, and 0.4 ml of water, sodium bicarbonate (19 mg, 0.23 mmol), and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol) were added thereto. The mixture was stirred at room temperature for 1 h. 20 ml of water was added thereto, followed by extraction 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 system C developing solvent to give the title product 10b (51 mg, yield 55.3%).

MS 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 10c

Compound 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 palladium on carbon (15 mg, 10% loading) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the crude title product 10C (13 mg).

MS 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

Compound 1b (10 mg, 18.8 μmol) was added to the reaction flask and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine, a solution of 10c (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 mixture was stirred in an ice bath for 30 min. 10 mL of water was added, followed by extraction 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 using developing solvent system B to give the title product 10d (16 mg, yield 97.8%).

MS 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-dioxo-2,3,9,10,13, 15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoropropionamide 10e

Compound 10d (16 mg, 18.4 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left to stand. Then, the supernatant was removed and the solid was aged; the 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.

MS 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-trofluoro-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 system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of 8 g (14 mg, 29.6 μmol) in 0.3 mL of N,N-dimethylformamide was added followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 μmol). The reaction mixture was stirred in an ice bath for 30 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 1 h to give compound 10. The reaction mixture 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 give the title products (2.7 mg, 2.6 mg).

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

Single configuration compound (with shorter retention time):

UPLC analysis: retention time: 1.18 min; purity: 91% (chromatographic column: ACQUITY UPLC BEHC18 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).

Single configuration compound (with longer retention time):

UPLC analysis: retention time: 1.23 min; purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 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-pentaazeicosyl)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)acetylamino)methoxy)cyclobutan-1-carboxylate 11b

Benzyl 1-hydroxycyclobutane carboxylate 11a (167 mg, 0.81 mmol, prepared as described in "Journal of Medicinal Chemistry, 2013, vol. 56, #13, p. 5541-5552") and 8b (150 mg, 0.41 mmol) were added to the reaction flask and 5 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of potassium tert-butoxide (92 mg, 0.82 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by the addition of 20 mL of ice water and extraction with ethyl acetate (5 mL×2) and chloroform (5 mL×5). The organic phases were combined and concentrated. The obtained residue was dissolved in 3 ml of dioxane, and 0.6 ml of water, sodium bicarbonate (41 mg, 0.48 mmol), and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) were added. The mixture was stirred at room temperature for 1 h. 20 ml of water was added, followed by extraction 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 obtained residue was purified by silica gel column chromatography using a system C developing solvent to give the title product 11b (37 mg, yield 17.6%).

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

Stage 2

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

Compound 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 palladium on carbon (15 mg, 10% loading) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture 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 used directly 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,12 H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclobutoxy)methyl)amino)-2-oxoethyl)carbamate 11d

Compound 1b (10 mg, 0.018 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine, a solution of compound 11c (13 mg, 0.031 mmol) in 0.5 ml of N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol). The reaction mixture was stirred in an ice bath for 40 min. 8 ml of water was added, followed by extraction 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 using developing solvent system A to give the title product 11d (19 mg, yield 73.9%).

MS 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 11e

Compound 11d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 4 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 11e (15 mg), which was used directly 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-pentaazeicosyl)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 system was flushed with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of 8 g (1.5 mg, 3.17 μmol) in 0.3 mL of N,N-dimethylformamide was added followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction mixture was stirred at room temperature for 30 min and concentrated to dryness with an oil pump to remove DMF (N,N-dimethylformamide), and the residue was dissolved in DCM (dichloromethane) and purified twice directly by thin layer chromatography (polarity of developing solvent: DCM/MeOH (MeOH - methanol) = 10/1) to give the title product 11 (1 mg, yield 28.8%).

MS 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-B

Stage 1

3-cyclopropyl-2-hydroxypropionic acid 12b

Compound 12a (0.5 g, 3.87 mmol, purchased from Adamas) was dissolved in 35 mL of a solvent mixture of water and acetic acid (V:V = 4:1) and the solution was cooled to 0-5 °C in an ice-water bath, followed by dropwise addition of 2 M aqueous sodium nitrite solution (0.53 g, 7.74 mmol). The reaction mixture was warmed to room temperature and stirred for 3 h. Solid sodium chloride was added to the reaction mixture to saturate the aqueous phase. The reaction mixture 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 mg, 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-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-B

To compound 1b (45 mg, 0.085 mmol) were added 1.5 ml of ethanol and 1.5 ml of N,N-dimethylformamide. The system was purged with argon three times and 0.1 ml of N-methylmorpholine was added dropwise. The reaction mixture was stirred until it became clear. 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 successively to the reaction mixture. After the addition, the reaction mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the obtained crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatography 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 products (7 mg, 15 mg).

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

Single configuration compound (with shorter retention time):

UPLC analysis: retention time: 1.345 min; purity: 72% (chromatographic column: ZORBAX Ecliphase Plus 18 1.8 μm 2.1×50 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).

Single configuration compound (with longer retention time):

UPLC analysis: retention time: 1.399 min; purity: 88% (chromatographic column: ZORBAX Ecliphase Plus 18 1.8 μm 2.1×50 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 as described in "Example 76 on page 147 of 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-B

Stage 1

Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

Compound 12b (200 mg, 1.54 mmol) was dissolved in 20 mL of acetonitrile and potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol) and tetrabutylammonium iodide (28 mg, 0.07 mmol) were added successively. The reaction mixture was stirred at room temperature for 48 h and 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 14a (140 g, 41.3% yield).

Stage 2

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

Compound 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to the reaction flask and 10 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (79 mg, 0.704 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 10 min followed by the addition of 20 mL of ice water and extraction 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 obtained residue was purified by silica gel column chromatography with developing solvent system C to give the title product 14b (50 mg, yield 26.8%).

MS 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

Compound 14B (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate and palladium on carbon (7 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times. The reaction mixture was stirred at room temperature for 1 h and 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.

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

Stage 4

(9H-fluoren-9-yl)methyl(2-((((3-cycloproyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

Compound 1b (22 mg, 42.38 μmol) was added to the reaction flask and 3 mL of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of triethylamine (4.3 mg, 42.49 μmol) and addition of crude 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 mixture was stirred in an ice bath for 40 min. 15 mL of water was added, followed by extraction 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%).

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

Stage 5

2-((2-aminoacetylamino)methoxy)-3-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)propanamide 14e

Compound 14d (29 mg, 33.9 μmol) was dissolved in 0.8 ml of dichloromethane, and 0.4 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left to stand. Then, the supernatant was removed; and the 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.

MS 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-B

Crude 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide. The system was flushed with argon three times and the solution was 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. Then the ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 1 h to give compound 14. The reaction mixture 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) to give the title products (2 mg, 2 mg).

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

Single configuration compound (with shorter retention time):

UPLC analysis: retention time: 1.18 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 µm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

Single configuration compound (with longer retention time):

UPLC analysis: retention time: 1.23 min; purity: 96% (chromatographic column: ACQUITY UPLC BEHC18 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

Compound 8b (500 mg, 1.35 mmol) was added to the reaction flask followed by the addition of 6 mL of tetrahydrofuran, benzyl 1-hydroxymethylcyclopropane-1-carboxylate 15A (233 mg, 1.13 mmol; prepared as described in "Example 22-2 on page 262 of the specification of patent application EP2862856A1"). The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of sodium hydride (54 mg, 1.35 mmol). Then, the ice bath was removed and the reaction mixture was warmed to room temperature, stirred for 40 minutes and cooled to 0 °C, followed by the addition of 20 ml of ice water and extraction 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, and 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 15b (15 mg, yield 2.5%).

MS 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

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

MS 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-b enzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

Compound 1b (10 mg, 0.021 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine, compound 15c (11 mg, 0.026 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol). After the addition, the reaction mixture was stirred at room temperature for 60 min. 10 ml of water was added, followed by extraction with ethyl acetate (5 ml×3). The organic phases were combined, 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 15d (19 mg, yield 87.0%).

MS 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 15e

Compound 15d (19 mg, 22.56 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated at 0 °C under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the 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.

MS 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 system was flushed with argon three times and the solution was cooled to 0-5 °C in an ice water bath followed by the addition of 8 g (15.8 mg, 33.4 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol). The reaction mixture was warmed to room temperature, stirred for 60 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 15 (2.5 mg, yield 10.3%).

MS 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-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

Stage 1

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

Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, purchased from Alfa) was dissolved in methanol (2 mL) and water (1 mL), and sodium hydroxide (126 mg, 3.15 mmol) was added. The reaction mixture 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 mixture 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 to dryness under reduced pressure; 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.

MS m/z (ESI, NEG (negative ion mass spectrometry)):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), and anhydrous potassium carbonate (1.09 g, 7.90 mmol), tetrabutylammonium iodide (29 mg, 78.51 mmol), and benzyl bromide (216 mg, 1.26 mmol) were added. The reaction mixture 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 using a system C developing solvent to give the title product 16c (112 mg, yield 32.1%).

MS 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

Compound 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to the reaction flask and 3 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (61 mg, 0.54 mmol). The reaction mixture was stirred in an ice bath for 10 min. 20 mL of ice water was added, followed by extraction 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 thereto. The reaction mixture was stirred at room temperature for 1 h. 20 ml of water was added, followed by extraction 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 system C developing solvent to give the title product 16d (24 mg, yield 16.7%).

MS 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

Compound 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 palladium on carbon (5 mg, 10% loading) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture 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 used directly 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

Compound 1b (7.5 mg, 0.014 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was 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 of N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6 mg, 0.026 mmol). The reaction mixture was stirred in an ice bath for 30 min. 10 ml of water was added, followed by extraction with ethyl acetate (10 ml×3). The organic phases were combined, 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 16f (10.6 mg, yield 87.8%).

MS 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)cyclobutan-1-carboxamide 16g

Compound 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 mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The residue was concentrated under reduced pressure to give the crude title product 16g (8 mg), which was directly used in the next step without purification.

MS m/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 and 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. The reaction mixture was stirred at room temperature for 30 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) to give the title product 16 (1.0 mg, yield 7.2%).

MS 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-nonaoxo-2,5,8,11,14-pentaa zatetratridec-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-decaoxaentriacontane-31-oate 17b

1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-ol 17a (0.34 g, 0.67 mmol, purchased from 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 mixture 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%).

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

Stage 2

tert-Butyl 29-hydroxy-3,6,9,12,15,18,21,24,27-nonoxanocosane-1-oate 17c

Compound 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran and palladium on carbon (110 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times and the reaction mixture was heated at 60 °C, stirred 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.

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

Stage 3

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

Compound 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, and diphenylphosphoryl azide (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added. The system was purged with argon three times, and the reaction mixture was stirred at room temperature for 2 h, then heated to 105 °C, reacted for 19 h, cooled to room temperature and concentrated. 20 mL of water was added, followed by extraction 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 obtained residue was purified by silica gel column chromatography using developing solvent system B to give the crude title product 17d (412 mg).

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

Stage 4

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

Compound 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran and palladium on carbon (58 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times. The reaction mixture was stirred at room temperature for 2 h and 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.

MS 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 system was purged with argon three times, and crude 17e (220 mg, 0.417 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. 15 mL of water was added, followed by extraction 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%).

MS 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

Compound 17f (122 mg, 0.163 mmol) was dissolved in 0.8 ml of dichloromethane and 0.4 ml of trifluoroacetic acid was added. The reaction mixture 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, then concentrated under reduced pressure; the procedures were repeated twice. Then 10 ml of toluene was added, and the resulting mixture was concentrated under reduced pressure, suspended three times with 10 ml of a solvent mixture of n-hexane:diethyl ether is 5:1 until pH reached 7, concentrated and oil pump dried to give the title product 17g (98 mg, yield 86.8%).

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

Stage 7

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

Compound 8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), and 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimine hydrochloride (115 mg, 0.60 mmol), and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added successively. After addition, the reaction mixture was stirred at room temperature for 1 h. 20 mL of water was added, and layers were formed 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 with developing solvent system C to give the title product 17h (124 mg, yield 55.4%).

MS 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-pentaazaptadecan-17-yl)oxy)cyclopropyl-1-carboxylate 17j

Compound 17h (39 mg, 69.6 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the 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 as described in "Example 7-12 on page 13 of the specification of patent application CN108853514A") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 μmol) were added thereto. The reaction mixture was stirred at room temperature for 1 h. 10 ml of water was added, followed by extraction 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%).

MS 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-pentaazaptadecan-17-yl)oxy)cyclopropan-1-carboxylic acid 17k

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

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[d e]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)carbamate 17l

Compound 1b (20 mg, 42.4 μmol) was added to the reaction flask, and 1 mL of 10% (v/v) methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine. The mixture was stirred until compound 1b was dissolved. Compound 17k (33 mg, 49.1 μmol) was dissolved in 1 mL of 10% (v/v) methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol). The reaction mixture was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added, and the resulting mixture was stirred for 5 minutes and left to stand to form 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 17l (37 mg, yield 80.2%).

MS 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-nonaoxo-2,5,8,11,14-pentaa zatetratridec-43-yl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

Compound 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 mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The reaction mixture was concentrated under reduced pressure and then dried using an oil pump. The obtained crude product was dissolved in 1 ml of N,N-dimethylformamide and compound 17g (11 mg, 15.92 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 μmol) were added. The system was purged with argon three times and the reaction mixture was stirred at room temperature, stirred for 30 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 17 (6 mg, 27.4% yield).

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

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.98 (d, 1H), 8.76 (s, 1H), 8.20 (br, 1H), 8.12-7.95 (m, 3H), 7.93-7.76 (m, 2H), 7.75-7.66 (m, 2H), 7.24 (s, 1H), 7.20-7.05 (m, 6H), 6.97 (s, 1H), 6.64 (br, 1H), 6.55 (d, 1H), 6.47 (s, 1H), 5.61-5.52 (m, 2H), 5.37 (s, 1H), 5.33-5.23 (m, 2H), 5.18 (s, 1H), 5.13 (s, 1H), 5.05 (s, 1H), 5.00 (s, 1H), 4.65-4.55 (m, 2H), 4.53-4.45 (m, 1H), 4.38-4.28 (m, 2H), 3.84 (s, 2H), 3.67 (d, 3H), 3.60-3.40 (m, 33H), 3.18 (d, 1H), 3.15-3.08 (m, 3H), 2.28 (s, 3H), 2.00-1.92 (m, 3H), 1.85 (s, 2H), 1.82-1.73 (m, 2H), 1.68-1.52 (m, 4H), 1.29-1.15 (m, 3H), 0.86-0.76 (m, 5H).

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

Compound 2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide (500 mg, 1.36 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 h and 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 (4.1 g) was purified by column chromatography on silica gel using C developing solvent system and further by chiral resolution 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 18c

Compound 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL) and compound 18a (2.0 g, 9.70 mmol) was added. The mixture was cooled to 0-5 °C in an ice-water bath and potassium tert-butoxide (1.89 g, 16.84 mmol) was added. The mixture was stirred in an ice-water bath for 10 min. Ethyl acetate (30 mL) and water (20 mL) were added and the resulting mixture was left to stand to form layers. The aqueous phase was extracted with chloroform (30 mL×5), the organic phases were combined and concentrated under reduced pressure. The obtained residue was dissolved in 1,4-dioxane (32 mL) and water (8 mL), and sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) were added. The resulting mixture was stirred at room temperature for 2 h. Water (30 mL) was added, followed by extraction 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 a C solvent system to give the title product 18c (1.3 g, yield 30.0%).

MS 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

Compound 18c (1.29 mg, 2.51 mmol) was dissolved in ethyl acetate (15 mL) and palladium on carbon (260 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 5 h 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.

MS 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-ester 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 mixture was stirred at room temperature for 1 h and concentrated under reduced pressure to remove the organic solvent. 20 mL of water was added, followed by extraction 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 developing solvent system C to give the title product 8e (810 mg, yield 61.1%).

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

Stage 5

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

Compound 18e (33 mg, 57.4 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 18f (21 mg), which was used directly 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-pentaazaicosan-20-oate 18g

Crude 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, and compound 17i (29 mg, 57.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 μmol) were added. The reaction mixture was stirred at room temperature for 1 h. 10 mL of water was added, followed by extraction 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%).

MS 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-pentaazaicosan-20-oic acid 18h

Compound 18g (37 mg, 44.3 μmol) was dissolved in 1.4 ml of 3% (v/v) dichloroacetic acid in dichloromethane, and the solution was cooled to 0-5 °C in an ice water bath, followed by the addition of triethylsilane (15.4 mg, 132.4 μmol). The reaction mixture was stirred in an ice bath for 3 hours. The reaction mixture was concentrated under reduced pressure in an ice bath to remove half of the organic solvent, and 5 ml of diethyl ether was added. The resulting mixture was warmed to room temperature naturally and 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 (24 mg, yield 79.1%).

MS 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

Compound 1b (30 mg, 63.6 μmol) was added to the reaction flask, and 1 mL of 10% (v/v) methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine. The mixture was stirred until compound 1b was dissolved. Compound 18H (65 mg, 94.8 μmol) was dissolved in 1 mL of 10% (v/v) methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol). The reaction mixture was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added, and the resulting mixture was stirred for 5 minutes and left to stand to form 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%).

MS 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-2,3,9,10,13,1 5-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropanamide 18j

Compound 18i (12 mg, 10.9 μmol) was dissolved in 0.6 ml of dichloromethane and 0.3 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 18j (10 mg), which was used directly in the next step without purification.

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

Stage 10

(1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10.13 -dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-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 and compound 17g (8.5 mg, 12.3 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol) were added. The reaction mixture was stirred at room temperature for 30 min and filtered. The filtrate 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 give the title product 18 (9.5 mg, 56.2% yield).

MS 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, 11H), 0.90-0.80 (m, 4H), 0.52-0.37 (m, 3H), 0.32-0.18 (m, 2H).

Example 1-19

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

Stage 1

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

Compound 18b (252 mg, 1.22 mmol) was added to the reaction flask and 4 mL of dichloromethane was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath followed by the addition of lithium tert-butoxide (98 mg, 1.22 mmol). The reaction mixture was stirred in an ice water bath for 15 min, it became clear, and 8b (300 mg, 814.3 μmol) was added. The reaction mixture was stirred in an ice water bath for 2.5 h. Water (10 mL) was added to separate the liquid. The aqueous phase was extracted with dichloromethane (8 mL×2), and the organic phases were combined and washed with water (10 mL×1) and saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to give a 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

Compound 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate and palladium on carbon (84 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times and the reaction mixture was stirred at room temperature for 3 h 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)carbonyldiimine hydrochloride (155 mg, 808.5 μmol), and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added sequentially. The reaction mixture 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 with 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

Compound 19c (60 mg, 104.4 μmol) was dissolved in 1 ml of dichloromethane and 0.5 ml of diethylamine was added. The reaction mixture 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; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 19d (21 mg), which was used directly 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-pentaazeicose-20-oate 19e

Crude 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, and compound 17i (52 mg, 103.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 μmol) were added. The reaction mixture was stirred at room temperature for 1 h. 10 mL of water was added, followed by extraction 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 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-pentaazeicose-20-oic acid 19f

Compound 19e (70 mg, 83.7 μmol) was dissolved in 2.5 ml of 3% (v/v) dichloroacetic acid in dichloromethane, and the solution was cooled to 0-5 °C in an ice water bath, followed by the addition of triethylsilane (29 mg, 249.4 μmol). The reaction mixture was stirred in an ice bath for 3 hours. The reaction mixture was concentrated under reduced pressure in an ice bath to remove half of the organic solvent, and 5 ml of diethyl ether was added. The resulting mixture was warmed to room temperature naturally and suspended to precipitate a white solid and filtered. The filter cake was collected and dried using 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

Compound 1b (30 mg, 63.6 μmol) was added to the reaction flask, and 1 mL of 10% (v/v) methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of triethylamine. The mixture was stirred until compound 1b was dissolved. Compound 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of 10% (v/v) methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol). The reaction mixture was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added, and the resulting mixture was stirred for 5 minutes and left to stand to form 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%).

MS 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-2,3,9,10,13,1 5-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 19h

Compound 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 mixture was stirred at room temperature for 1.6 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the 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-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

Crude 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide and compound 17g (2.9 mg, 4.2 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol) were added. The reaction mixture was stirred at room temperature for 40 min and filtered. The filtrate 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 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, 11H), 0.94-0.78 (m, 5H), 0.52-0.35 (m, 3H).

Example 1-20 (reference example)

The title compound 20 was prepared as described in "Example 58 on page 163 of patent specification CN104755494A".

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

The sequence of trastuzumab is shown below:

Light chain

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:1

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:2

The sequence of pertuzumab is shown below:

Light chain

DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:3

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:4

Below is the B7H3 sequence of the 1F9DS antibody:

Light chain

DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKL TVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

SEQ ID NO:5

Heavy chain

QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQG ALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:6

Example 1-21. ADC-1

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 μL, 0.82 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

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 solution was added to the above 2.0 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product of ADC-1 FADC-1 of the general formula in PBS buffer (5.0 mg/mL, 1.1 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 5.09.

Example 1-22. ADC-2

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

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

The average value calculated by UV-HPLC: n is 7.39.

Example 1-23. ADC-3

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO and the solution was added to the above 2.0 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product of ADC-3 FADC-3 of the general formula in PBS buffer (5.24 mg/mL, 1.1 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 7.36.

Example 1-24. ADC-4

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 3.74 mL, 13.38 mg/mL, 0.338 μmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 6.7 mg/mL, and 1.3 mL of the solution was collected for use in the next step.

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 solution was added to the above 1.3 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-4 FADC-4A of the general formula in PBS buffer (1.72 mg/mL, 2.36 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 7.39.

Example 1-25. ADC-5

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 3.0 mL, 6.70 mg/mL, 0.136 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and 0.614 mL of the solution was collected for use in the next step.

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 solution was added to the above 0.614 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-5 FADC-4A of the general formula in PBS buffer (3.08 mg/mL, 0.82 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 3.16.

Example 1-26. ADC-6

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 3.74 mL, 13.38 mg/mL, 0.338 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 6.7 mg/mL, and 0.75 mL of the solution was collected for use in the next step.

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 solution was added to the above 0.75 mL solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-6 FADC-4B of the general formula in PBS buffer (1.78 mg/mL, 1.78 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 3.94.

Example 1-27. ADC-7

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the pertuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 1.0 mL of the solution was collected for use in the next step.

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

The average value calculated by UV-HPLC: n is 6.91.

Example 1-28. ADC-8

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the pertuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 1.6 mL of the solution was collected for use in the next step.

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 solution was added to the above 1.6 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-8 FADC-8 of the general formula in PBS buffer (2.14 mg/mL, 2.31 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 6.58.

Example 1-29. ADC-9

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the pertuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) at 37 °C. The reaction mixture was shaken in a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath and diluted to 5.0 mg/mL, and 0.8 mL of the solution was collected for use in the next step.

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 solution was added to the above 0.8 mL of solution. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-9 FADC-9A of the general formula in PBS buffer (2.27 mg/mL, 1.11 mL), which was then stored at 4 °C.

The average value calculated by UV-HPLC: n is 3.16.

Example 1-30. ADC-10

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.574 mL, 38.78 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-10 FADC-10 of the general formula in PBS buffer (5.48 mg/mL, 1.03 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.25.

Example 1-31. ADC-11

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.646 mL, 43.64 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath.

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

The average value calculated by the UV-Vis: n method is 7.03.

Example 1-32. ADC-12

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.726 mL, 49.05 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-12 FADC-12 of the general formula in PBS buffer (3.34 mg/mL, 1.45 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.93.

Example 1-33. ADC-13

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.287 mL, 19.39 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-13 FADC-13 of the general formula in PBS buffer (2.37 mg/mL, 0.88 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.53.

Example 1-34. ADC-14

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.592 mL, 40.0 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-14 FADC-14 of the general formula in PBS buffer (0.30 mg/mL, 12.0 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.61.

Example 1-35. ADC-15

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.592 mL, 40.0 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-15 FADC-15 of the general formula in PBS buffer (0.32 mg/mL, 11.8 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.89.

Example 1-36. ADC-16

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.53 mL, 35.8 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath.

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

The average value calculated by the UV-Vis: n method is 7.43.

Example 1-37. ADC-17

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 2.0 mL, 135.12 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-17 FADC-4A of the general formula in PBS buffer (1.32 mg/mL, 12.0 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 5.42.

Example 1-38. ADC-18 (Reference Example)

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 1.5 mL, 101.3 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-18 FADC-18 of the general formula in PBS buffer (0.79 mg/mL, 13.0 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.23.

Example 1-39. ADC-19

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 1.36 mL, 91.9 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product of ADC-19 FADC-4A of the general formula in PBS buffer (0.73 mg/mL, 13.0 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.26.

Example 1-40. ADC-20

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 1.5 mL, 101.3 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath.

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

The average value calculated by the UV-Vis: n method is 7.43.

Example 1-41. ADC-21 (Reference Example)

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 1.86 mL, 125.4 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-21 FADC-18 of the general formula in PBS buffer (2.91 mg/mL, 4.44 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.23.

Example 1-42. ADC-22

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 1.88 mL, 127.2 nmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-22 FADC-4A of the general formula in PBS buffer (3.56 mg/mL, 3.98 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.79.

Example 1-43. ADC-23 (reference example)

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 μL, 118.9 μmol) was added to trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 345 mL, 23.31 μmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3.5 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath.

Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL MeCN (acetonitrile) and 3.56 mL DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was purified by desalting through ultrafiltration membranes with 2% (v/v) MeCN and 1% (v/v) DMSO-containing aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5) and aqueous succinic acid buffer (0.01 M aqueous succinic acid buffer at pH 5.3) sequentially. Sucrose was then added at a concentration of 60 mg/mL and tween 20 at a concentration of 0.2 mg/mL. The mixture was bottled and lyophilized to obtain a lyophilized powder sample of the illustrative product ADC-23 of the general formula FADC-18, which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.05.

Example 1-44. ADC-24

The resulting aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol) was added to the trastuzumab antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 332 mL, 22.43 μmol) at 37 °C. The reaction mixture was shaken on a shaking water bath at 37 °C for 3.5 h before stopping the reaction. The reaction mixture was cooled to 25 °C in a water bath.

Compound 9 - Compound 9A with shorter retention time (241 mg, 224 μmol) was dissolved in 13.76 mL MeCN and 6.88 mL DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was purified by desalting through ultrafiltration membranes with 4% (v/v) MeCN and 2% (v/v) DMSO-containing aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5) and aqueous succinic acid buffer (0.01 M aqueous succinic acid buffer at pH 5.3) sequentially. Sucrose was then added at a concentration of 60 mg/mL and tween 20 at a concentration of 0.2 mg/mL. The mixture was bottled and lyophilized to obtain a lyophilized powder sample of the illustrative product ADC-24 of the general formula FADC-4A, which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.07.

Example 1-45. ADC-25

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 2.14 mL, 144.60 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-25 FADC-25 of the general formula in PBS buffer (1.28 mg/mL, 13.0 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.87.

Example 1-46. ADC-26 (Reference Example)

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on a water bath.

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

The average value calculated by the UV-Vis: n method is 6.15.

Example 1-47. ADC-27

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product of ADC-27 FADC-25 of the general formula in PBS buffer (1.94 mg/mL, 3.5 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 6.11.

Example 1-48. ADC-28 (reference example)

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 2.36 mL, 159.47 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on a water bath.

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

The average value calculated by the UV-Vis: n method is 7.46.

Example 1-49. ADC-29

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.80 mL, 50.06 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on 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 solution was added to the above reaction mixture. The reaction mixture was shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5 containing 0.001 M EDTA) to obtain an exemplary product ADC-29 FADC-25 of the general formula in PBS buffer (2.63 mg/mL, 2.4 mL), which was then stored at 4 °C.

The average value calculated by the UV-Vis: n method is 7.24.

Example 1-50. ADC-30 (reference example)

To the B7H3 antibody 1F9DS antibody in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.86 mL, 58.4 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on a water bath.

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

The average value calculated by the UV-Vis: n method is 6.15.

Example 1-51. ADC-31

To the B7H3 antibody 1F9DS antibody in PBS aqueous buffer (0.05 M PBS aqueous buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) at 37 °C was added the obtained aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a shaking water bath at 37 °C for 3 h before stopping the reaction. The reaction mixture was cooled to 25 °C on a water bath.

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

The average value calculated by the UV-Vis: n method is 6.33.

Example 1-52. ADC-32

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

Compound 9 - Compound 9-A with a shorter retention time (406.2 mg, 378.17 μmol) was dissolved in 9.98 mL of DMSO to obtain a DMSO solution of Compound 9-A. 23.42 mL of DMSO was pre-added to the above solution of Intermediate I, and then the above solution of Compound 9-A in DMSO was added to the solution of Intermediate I to which DMSO was added in advance. The reaction mixture was stirred in a water bath at 25 °C for 1 h, quenched with cysteine, and filtered. At 25 °C, the reaction mixture was buffer exchanged by 10-fold and 16-fold volumetric equal-volume ultrafiltration through an ultrafiltration membrane (30 kDa) with 10% (v/v) DMSO-containing aqueous buffer containing 20 mM histidine hydrochloric acid and 2.5 mM EDTA (pH = 6.0) and an aqueous buffer containing 10 mM histidine hydrochloric acid (pH is 5.5), sequentially to remove small molecules and residual solvents, and a typical product ADC-32 of the general formula FADC-4A was obtained.

The mean value calculated by HIC:n is 6.0.

Example 1-53. ADC-33

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

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

The mean value calculated by HIC: n is 7.15.

Drug loading analysis of ADC stock solution

The purpose and principle of the experiment

ADC is a cross-linked drug with an antibody. Its mechanism of treating diseases is to transfer toxin molecules into cells depending on the targeting ability of the antibody and kill the cells. The drug loading plays a decisive role in the efficacy of the drug. The drug loading of the ADC stock solution was determined by UV-Vis spectrophotometry or hydrophobic interaction chromatography (HIC).

1. Spectroscopy in the visible and ultraviolet spectral regions

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

Calculation: The loading of the ADC stock solution was determined using visible-ultraviolet spectroscopy (device: Thermo nanodrop2000 ultraviolet spectrophotometer), based on the principle that the total absorbance value of the ADC stock solution at a certain wavelength is the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength, namely:

(1) A _{280 nm} = ε _mab-280 bC _mab + ε _Drug-280 bC _Drug (mab - monoclonal antibody, Drug - medicinal product)

ε _Drug-280 : the mean molar extinction coefficient of the drug at 280 nm was 5100;

C _Drug : drug concentration;

ε _mab-280 : The mean molar extinction coefficient of trastuzumab stock solution or pertuzumab stock solution at 280 nm was 214,600;

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

b: The optical path length is 1 cm.

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

(2) A _{370 nm} = ε _mab-370 bC _mab + ε _Drug-370 bC _Drug

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

C _Drug : drug concentration;

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

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

b: The optical path length is 1 cm.

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

Drug loading n = C _Drug /C _mab .

2. Hydrophobic interaction chromatography

2.1 HPLC analysis was carried out under the following measurement conditions:

HPLC System: Agilent HPLC High Performance Liquid Chromatography System

Detector: DAD (Photodiode Array Detector) (Measuring wavelength: 280nm)

Chromatography column: TSKgel Butyl-NPR (4.6 mm×100 mm, 2.5 μm)

Column temperature: 30 °C

[Flow Rate]: 0.5ml/min

Sample chamber temperature: 4°C

Mobile phase A: aqueous solution at pH 7.00 containing 1.5 M ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and 20 mM disodium phosphate (Na ₂ HPO ₄ ).

Mobile phase B: a mixed solution with pH 7.00 containing 75% 20 mM disodium phosphate (Na ₂ HPO ₄ ) and 25% isopropanol.

Gradient program: from 0.0% B to 0.0% B (from 0 min to 3.0 min), from 0.0% B to 100.0% B (from 3.0 min to 30.0 min), from 100.0% B to

100.0% B (from 30.0 min to 33.0 min), from 0.0% B to 0.0% B (from 33.1 min to 40.0 min),

Injection volume: 50 µg

2.2 Data analysis

Based on the current data, due to the characteristics of the column, based on the difference in salt concentrations, the antibody-drug conjugate was eluted in ascending order according to the amount of bound drugs. Therefore, the distribution of the amount of conjugated drugs was obtained by measuring the area of each peak. According to the elution order, the peaks were D0 (antibody without binding to the drug linker), D2, D4, D6 and D8. The drug loading DAR (n) was calculated as follows:

Table 1. Calculation of drug loading by hydrophobic interaction chromatography Name Number of related drugs Peak load area in percent D0 0 Peak area D0 × number of bound drugs D2 2 Peak area D2 × number of bound drugs D4 4 Peak area D4 × number of bound drugs D6 6 Peak area D6 × number of bound drugs D8 8 Peak area D8 × number of bound drugs

The drug loading n is equal to Σ (area of peak loading)/100

Biological assessment

Test Example 1-1: In vitro tumor cell proliferation inhibition test by the compounds described in this document

I. Purpose

This experiment is designed to determine 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 (American Type Culture Collection), Cat. No. HTB-30). The cells were treated in vitro with the compound at different concentrations. After 6 days of culture, cell proliferation was determined using a CTG reagent (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

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

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

2. Cell preparation: U87MG and SK-BR-3 cells growing in log phase were collected, washed once with PBS (phosphate-buffered saline, 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 complete digestion, the cells were eluted with 10-15 ml cell culture medium; the eluate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; then the cells were resuspended in 10-20 ml cell culture medium to form single-cell suspensions.

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

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

Low molecular weight compounds were in an initial concentration of 500 nM. The dosage form was prepared as follows.

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 column through the eleventh column. The samples in the first row (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 until 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 an initial concentration of 10 nM or 500 nM as follows.

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

5. Test samples prepared at different concentrations (20 µl) were added to a culture plate, with two duplicate wells set up for each sample. The plate was incubated in an incubator (37 °C, 5% CO ₂ ) for 6 days.

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

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

III. Data Analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results of the experiment are presented in Table 2 below.

Table 2. IC ₅₀ values of low molecular weight fragments of the present invention in inhibiting in vitro proliferation of SK-BR-3 cells and U87 cells Connection No. IC ₅₀ (nM) SK-BR-3 U87 1 0.12 0.23 2 - 2-B with shorter retention time 0.33 0.86 2 - 2-A with a longer retention time 8.11 2.31 3 - with shorter retention time 0.36 0.83 3 - with a longer retention time 1.67 2.98 4 1.9 / 5 / 4.81 6 / 1.83 7 / 1.95

Conclusion: The low molecular weight fragments of 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 inhibition test of HER2-targeted tumor cell proliferation using the antibody-drug conjugates of the present invention

This experiment was designed to determine the inhibitory activity of the HER2-targeting antibody drug conjugates of the present invention against the 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 determined using CTG reagents and the in vitro activity of the compound was assessed according to the IC ₅₀ value.

The test method of Test Example 1 was used. SK-BR-3 and MDA-MB-468 cells were used in the tests, and the cell culture media were 10% FBS-containing McCoy 5A medium (Gibco, Cat. No. 16600-108), 10% FBS-containing EMEM medium (GE, Cat. No. SH30024.01), and 10% FBS-containing L-15 medium (ThermoFisher, Cat. No. 11415-114). 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 cell culture medium, respectively, and the density-adjusted cell suspensions were mixed well and added to 96-well cell culture plates at 180 μL/well. The corresponding compounds were tested, and the results are shown in Table 3 below.

Table 3. IC ₅₀ values of antibody-drug conjugates of the present invention in inhibiting in vitro proliferation of HER2-targeted tumor cells Connection No. IC ₅₀ (nM) SK-BR-3 MDA-MB-468 ADC-3 0.43 more than 50 ADC-4 0.30 more than 50 ADC-6 0.48 more than 50 ADC-7 0.14 more than 50 ADC-9 0.95 more than 50 ADC-10 1.36 more than 50 ADC-11 0.73 more than 50 ADC-12 0.82 more than 50 ADC-13 0.47 more than 50 ADC-14 0.53 more than 50 ADC-15 0.38 more than 50 ADC-16 0.49 more than 50 ADC-17 0.37 more than 50

Conclusion: The HER2-targeting antibody-drug conjugates of the present invention showed significant inhibitory activity against the proliferation of HER2-positive SK-BR-3 cells, while showing weak inhibitory activity against the proliferation of HER2-negative MDA-MB-468 cells, demonstrating good selectivity.

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

ADC-19, ADC-18, and ADC-20 samples, human plasma, monkey plasma (Shanghai Medicilon Inc.), and 1% BSA (bovine serum albumin) (Sigma) in PBS (Sangon Biotech (Shanghai)) were filtered through a 0.22 μm filter for sterilization. Each of ADC-19, ADC-18, and ADC-20 was added to sterile plasma or 1% BSA in PBS at a final concentration of 200 μg/mL. The resulting mixture was incubated in a cell incubator at 37 °C. The day of incubation was defined as day 0. Samples were collected at days 7, 14, and 21 and tested for free toxin.

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

The results showed that: ADC-19 was reasonably stable in both human and monkey plasma as well as in 1% BSA 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 free toxin release rates of 14.5% and 8.10% at the highest level, respectively. It was relatively stable in 1% BSA in PBS, see Fig. 1B.

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

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

I. Purpose

Using nu/nu nude mice as experimental animals, Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 were administered by intraperitoneal injection and then their efficacy was assessed in nude mice bearing trastuzumab (Herceptin)-resistant human breast cancer tumor line JIMT-1 xenograft.

II. Test medicinal products and materials

1. Test drugs

T-DM1 (received with reference to US20050169933)

ADC-21: 3 mg/kg

ADC-21: 10 mg/kg

ADC-24: 3 mg/kg

ADC-24: 10 mg/kg

Blank control: PBS

2. Method of preparation: all obtained by dilution in PBS.

3. Test animals

nu/nu nude mice purchased from Beijing Vital River.

III. Method

Mice were subcutaneously inoculated in the right flank with JIMT-1 cells (Nanjing Cobioer) (5× ¹⁰⁶ cells/mouse, with 50% artificial basement membrane). After 8 days, tumors grew to a size of 203.09±11.94 ^mm3 , and animals were randomized into 6 groups of 8 (d1).

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

Excel 2003 statistical software was used. Mean values were calculated as avg; SD (standard deviation) values were calculated as STDEV (square root of the sample variance); SEM (standard error of the mean) values were calculated as STDEV/SQRT (square root); and the difference in P value between groups was calculated as TTEST.

Tumor volume (V) was calculated as: V = 1/2 × L _length × L _width 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 of the blank group (vehicle, PBS) and the experimental groups, respectively, at the end of the experiment.

IV. 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). Under the same dosage condition, the tumor inhibition effect of ADC-24 was significantly better than that of ADC-21.

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

I. Purpose

Using nu/nu nude mice as experimental animals, Her2-ADC antibodies ADC-21 and ADC-22 were administered by intraperitoneal injection and then their efficacy was assessed in nude mice bearing human breast cancer xenograft SK-BR-3 cells.

II. Test medicinal products and materials

1. Test drugs

ADC-21: 1 mg/kg

ADC-21: 6 mg/kg

ADC-22: 1 mg/kg

ADC-22: 6 mg/kg

Blank control: PBS.

2. Method of preparation: all obtained by dilution in PBS.

3. Test animals

nu/nu nude mice purchased from Beijing Vital River.

III. Method

Mice were subcutaneously inoculated in the right flank with SK-BR-3 cells (ATCC) (5× ¹⁰⁶ cells/mouse, with 50% artificial basement membrane). After 20 days, tumors grew to a size of 153.34±11.73 ^mm3 , and animals were randomized into 5 groups of 8 (d0).

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

Excel 2003 statistical 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 difference between groups was calculated as TTEST.

Tumor volume (V) was calculated as: V = 1/2 × L _length × L _width 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 of the blank control and experimental groups, respectively, at the end of the experiment.

IV. 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% and had a significant difference compared with 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). Under the same dosage condition of 6 mg/kg, the tumor inhibition effect of ADC-22 was significantly better than that of ADC-21.

Test Example 1-6: Plasma Stability

ADC-25 sample was mixed well with each of human plasma, monkey plasma, and 1% BSA in PBS at a final concentration of 100 μg/mL, and then the mixtures were filter sterilized and incubated in a water bath at 37 °C. The day of incubation was defined as day 0. Samples were collected on days 7, 14, and 21 and tested for free toxin.

Samples were collected at different time points, allowed to cool to room temperature, and mixed well using a vortex mixer. 25 μL of each sample was added to a 96-well plate, followed by the addition of 50 μL of the internal standard working solution (100 ng/mL camptothecin in acetonitrile) and 150 μL of acetonitrile. The resulting mixtures were vortexed for 5 min and centrifuged at 4000 rpm for 10 min. 5 μL of the supernatant was collected for LC/MS/MS analysis.

The results showed that: ADC-25 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% at the highest level, and was generally stable at day 14, see Fig. 4.

Test Case 1-7: Evaluation of the efficacy of ADC against human brain astrocytoma tumor U87MG xenografted into nude mice.

I. Purpose

Using BALB/cA nude mice as experimental animals, this experiment evaluated the efficacy of the ADC compounds of the present invention against human U87MG brain astrocytoma tumor xenografted into nude mice.

II. Test medicinal products and materials

1. Test drugs

ADC-27 (3 mg/kg)

ADC-26 (3 mg/kg)

Blank: PBS buffer pH 7.4.

2. Method of preparation: PBS buffer with pH 7.4.

3. Test animals

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

III. Method

Six- to seven-week-old laboratory 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 grouped (D0) with 8 animals per group, injected intraperitoneally once a week for a total of 3 times, and the tumor volumes and body weights 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 of the control (blank) and experimental groups, respectively, at the end of the experiment.

IV. Results

Intraperitoneal (i.p.) administration 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 demonstrated higher antitumor efficacy than ADC-26.

The weight of all animals in each group was normal during the administration process, and the ADCs had no obvious toxicity or side effect. The results are shown in Table 4 and Fig. 5. The detected antibodies could effectively inhibit the growth of U87MG xenograft tumor in tumor-bearing nude mice and showed dose dependence.

Table 4. Efficacy of administered antibodies against human brain astrocytoma tumors U87MG xenografted into nude mice (D22) Group Average tumor volume ( ^mm3 ) Relative tumor volume Tumor inhibition rate (%) Day 0 SEM (scanning electron microscopy) Day 22 SEM Day 22 SEM Day 22 Idle control 167.06 17.74 2906.96 327.6 17.76 1.63 - ADC-27 3 mg/kg 167.07 16.06 1172.48 80.27 7.55 0.95 63.3*** ADC-26 3 mg/kg 167.73 17.63 1561,03 303.37 8.83 1.17 49.1*** *** denotes P < 0.001

Test Example 1-8: Evaluation of the efficacy of ADC against Detroit 562 human metastatic pharyngeal cancer tumor cells with pleural effusion xenografted into nude mice.

I. Purpose

In this experiment, the efficacy of the ADC compounds of the present invention against Detroit 562 human metastatic pharyngeal cancer tumor cells with pleural effusion xenografted into nude mice was assessed.

II. Test medicinal products and materials

1. Test drugs

ADC-29 (3 mg/kg)

ADC-28 (3 mg/kg)

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

2. Method of preparation: all obtained by dilution in PBS.

3. Test animals

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

III. Method

Six to seven-week-old female laboratory nude BALB/cA mice were inoculated subcutaneously with Detroit 562 human metastatic pharyngeal carcinoma cells from pleural effusion (ATCC Cat. No. ATCC ^® CCL-138™). On the tenth day after cell inoculation, animals were randomly grouped (D0) with 8 animals per group, injected intraperitoneally once per week for a total of 3 times, and tumor volumes and body weights were measured 2-3 times per week and 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 of the control (negative) and experimental groups, respectively, at the end of the experiment.

IV. Results

The drugs were administered by intraperitoneal injection once a week, and a total of 3 injections were performed. On day 28 of observation, the tumor inhibition rates of the tested ADCs ADC-29 3 mg/kg (3 mg/kg) and ADC-28 3 mg/kg mg/kg (3 mg/kg) reached 72.27% (P < 0.001) and 56.2% (P < 0.001), respectively. ADC-29 demonstrated higher antitumor efficacy than ADC-28.

The weight of all animals in each group was normal during the administration process, and the ADCs had no obvious toxicity or side effect. The results are shown in Table 5 and Fig. 6. The detected antibodies could effectively inhibit the growth of Detroit 562 xenograft tumor in tumor-bearing nude mice and showed dose dependence.

Table 5. Efficacy of administered antibodies against Detroit 562 xenograft tumors in tumor-bearing nude mice (D28) Group Average tumor volume ( ^mm3 ) Average tumor volume ( ^mm3 ) Relative tumor volume Tumor inhibition rate (%)
Day 28 Day 0 SEM Day 28 SEM Day 28 SEM Negative control 182.70 6.79 1317.99 223.20 7.47 1.46 - ADC-29 mg/kg 182.59 6.50 381.48 105.76 2.07 0.58 72.27*** ADC-28 3 mg/kg 182.57 6.92 578.07 160.13 3.43 1.09 56.2*** *** denotes P < 0.001

Test Case 1-9: Evaluation of the drug efficacy in U87-MG tumor-bearing mice

I. Purpose

Using nude Balb/c mice as experimental animals, the efficacy of the B7H3 antibody-drug conjugate after intraperitoneal injection was evaluated in a model of human glioblastoma U87MG tumor cells xenografted into mice.

II. Test medicinal products and materials

1. Test drugs

ADC-30 1 mg/kg

ADC-30 3 mg/kg

ADC-31 1 mg/kg

ADC-31 3 mg/kg

Blank control: PBS

2. Method of preparation: all obtained by dilution in PBS.

3. Test animals

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

III. Method

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 cells/mouse). After 14 days, the tumors grew to a size of 167.49 ^mm3 , and the animals were randomized into 5 groups of 8 (d1).

The drug was administered by intraperitoneal injection once a week for a total of 3 injections. Tumor volumes and body weights were measured twice a week and the results were recorded.

Excel 2003 statistical 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 difference between groups was calculated as TTEST.

Tumor volume (V) was calculated as: V = 1/2 × L _length × L _width 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 of the blank control group (vehicle) and the experimental groups, respectively, at the end of the experiment.

IV. Results

The experimental results are shown in Fig. 7. The drugs were administered by intraperitoneal injection once a week, and a total of 3 injections were performed. On the 18th day of observation, the tumor inhibition rates of the test ADCs ADC-30 1 mg/kg, ADC-30 3 mg/kg, ADC-31 1 mg/kg and ADC-31 3 mg/kg reached 0.31%, 45.23% (P < 0.0001), 39.22% (P < 0.01) and 80.24% (P < 0.0001), respectively. Under the same dosage condition, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

II. Compositions

The equipment used in obtaining and determining the composition and the method of calculating the results are shown below.

SEC molecular size exclusion chromatography:

This is a method of analyzing the separation of a solute by the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

SEC% (SEC percentage of monomer content) = A monomer/A total × 100% (A monomer is the peak area of the main monomer peak in the sample, and A total is the sum of the areas of all peaks).

SEC measurement instrument: Agilent 1260; column: water, XBrige BEH200Å SEC (300 × 7.8 mm 3.5 µm).

Capillary gel electrophoresis CE:

It is a method of moving gel into a capillary as a supporting medium for electrophoresis and separation according to the molecular weight of the sample under a certain voltage.

Reduced purity percentage CE = A main peak/A total × 100% (A main peak is the area of the light chain main peak + the area of the heavy chain main peak, and A total is the sum of all peak areas).

CE measuring device: Beckman plus800 model

Turbidity measurement:

The degree to which light does not pass through a water layer indicates the ability of the water layer to scatter and absorb light. It is related not only to the suspended solids content but also to the composition, size, and shape of the particles, as well as the reflectance characteristics of their surfaces. When comparing the absorbance values of the same protein sample at the same concentration at the same wavelength (within the near ultraviolet and visible wavelength ranges), the higher the absorbance value, the greater the turbidity, and the more protein molecules in the sample are prone to aggregation. The measuring instrument was a multi-plate reader (Molecular Devices M5). The same volume of samples were added to a 96-well plate, and the absorbance values were read.

Measurement of osmotic pressure:

The freezing point method was used to measure the osmotic pressure. The freezing point of the solution was measured by a highly sensitive temperature-sensitive element based on the proportional relationship between the freezing point depression value and the molar concentration of the solution, and then converted into osmotic pressure through electrical quantity. Instrument manufacturer: Loser, model: OM815.

Determination of protein concentration:

Since the toxin in the antibody-drug conjugate absorbed light at 280 nm, the protein concentration was adjusted using the following formulas:

A ₂₈₀ = C _d × ε _280d + C _mab × ε _280mab

A ₃₇₀ = C _d × ε _370d ,

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

Protein concentration measuring instrument: UV-visible spectrophotometer; Model: Nano Drop oneC; Optical path length: 1mm.

As an example, the molecular weight of the naked antibody is 145.181 kDa and the molecular weight of the toxin is 1074 Da; if the average DAR value is 5.7, the calculated molecular weight of the ADC will be 151.317 kDa, which is 1.042 times that of the naked antibody. As an example, if the ADC DAR value is 5.7 and the protein concentration of the naked antibody is 20.00 mg/mL, the ADC concentration will be 20.84 mg/mL.

Example 2-1 Screening of buffer systems and pH values for drugs

20 mg/mL (protein concentration based on naked antibody) of ADC-33-based preparation (containing no saccharide, surfactant or other buffers except the following buffer systems) was prepared using the following buffer systems:

1) 10 mM sodium histidine acetate, pH 5.0

2) 10 mM sodium histidine acetate, pH 5.5

3) 10 mM sodium histidine acetate, pH 6.0

4) 10 mM sodium histidine acetate, pH 6.5

5) 10 mM histidine-histidine hydrochloride, pH 5.5

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

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

Samples were collected for high temperature stability studies (40°C) and shake studies (25°C, 300 rpm). The shake sample contained 0.1 mg/mL polysorbate 80 (PS80) and the remaining samples contained 0.4 mg/mL PS80. Each formulation was filtered and the container was filled, stoppered and capped. Samples were subjected to the forced degradation experiments described above and analyzed for appearance, SEC, and turbidity.

The results are presented in Table 6. The results show that where sodium histidine acetate was used as the buffer, the appearance of the samples gradually deteriorated, the turbidity increased, and the SEC% monomer proportion decreased as the pH value increased in the range of pH 5.0-6.5. At pH 5.5, the sample containing 10 mM sodium histidine acetate and the sample containing 10 mM histidine hydrochloride were similar in appearance and SEC% and were superior to the samples containing 10 mM succinic acid sodium succinate or 10 mM citric acid sodium citrate. Unexpectedly, the preparation containing 10 mM succinic acid sodium succinate was significantly superior in turbidity to the other preparations after the shaking test.

Table 6. Results of pH determination and stability of the buffer system No. Terms and Conditions Appearance Turbidity A550 SEC% 1 D0 Clean and clear 0.051 95.9 40 °C D12 Slightly opalescent 0.058 92.0 Shaking D12 Slightly opalescent 0.055 95.0 2 D0 Clean and clear 0,050 95.8 40 °C D12 Slightly opalescent 0.061 91.6 Shaking D12 Slightly opalescent 0.056 93.3 3 D0 Clean and clear 0.051 95.4 40 °C D12 Slightly opalescent 0.061 91.0 Shaking D12 Slightly opalescent 0,060 91.3 4 D0 Clean and clear 0,050 94.4 40 °C D12 Large amount of clouding particles 0.085 89.3 Shaking D12 Slightly opalescent 0.058 87.4 5 D0 Clean and clear 0.051 95.5 40 °C D12 Slightly opalescent 0.065 90.9 Shaking D12 Slightly opalescent 0,060 93.1 6 D0 Clean and clear 0.053 95.7 40 °C D12 Slightly opalescent fine particles 0.055 89.7 Shaking D12 Slightly opalescent 0.053 91.5 7 D0 Clean and clear 0.057 95.5 40 °C D12 Opalescent 0.063 88.8 Shaking D12 Opalescent 0.059 89.3

Note: In the table, "D" stands for day; for example, D12 stands for day 12, and D0 stands for the beginning of the experiment; the same applies below.

Example 2-2. Screening of surfactant types

20 mg/mL preparations (protein concentration based on naked antibody) of ADC-32 containing different polysorbate surfactants were prepared, each containing 10 mM succinic acid-sodium succinate buffer (pH 5.0) and 80 mg/mL sucrose. Each preparation was filtered and the container was filled with it, stoppered and capped. Samples were subjected to high temperature (40 °C) stability testing, shaking testing (25 °C, 300 rpm), appearance testing, SEC and recovered CE.

The results are shown in Table 7. There were no significant differences in appearance, SEC, and recovered CE between the PS20 (polysorbate 20)-containing formulations and PS80-containing formulations under different conditions, indicating that both PS20 and PS80 were able to effectively stabilize ADC-32.

Table 7. Results of screening of types of polysorbates Types of polysorbates Terms and Conditions Appearance SEC% Reduced CE% PS80
0-0.2 mg/ml D0 Clean and clear 99.1 98.7 Shaking D15 Clean and clear 97.2 98.4 40 °C D15 Clean and clear 95.4 98.3 PS20
0-0.2 mg/ml D0 Clean and clear 99.0 98.7 Shaking D15 Clean and clear 97.7 98.7 40 °C D15 Clean and clear 95.4 98.2

Examples 2-3. Screening of surfactant concentrations

Samples containing 0, 0.1, 0.2, 0.4, and 0.6 mg/mL PS80 were prepared. Each sample also contained 10 mM succinic acid-sodium succinate (pH 5.0), 80 mg/mL sucrose, and 20 mg/mL (protein concentration based on naked antibody) ADC-32. Samples were filtered to remove bacteria and particles and diluted with 0.9% NaCl such that the ADC-32 concentration was 0.2 mg/mL. Dilutions were examined for appearance and the presence of insoluble particulates.

The results are shown in Table 8. When the concentration of PS80 in the preparation was 0-0.2 mg/mL, as the concentration increased, the increase in the number of insoluble microparticles in the dilution became smaller, and the visible particles were fewer and fewer. Where the concentration was 0.2-0.6 mg/mL, there was no difference in insoluble microparticles between the dilutions and the appearance was good.

Table 8. Results of PS80 concentration screening Concentration of PS80 Time Appearance Insoluble microparticles/100 ml 2 µm or more 10 µm or more 25 µm or more 0 mg/ml 0 h Clean and clear, single particles 8892 458 33 4 °C 24 h Individual particles 39408 4317 100 0.1 mg/ml PS80 0 h Clean and clear, single particles 5267 525 17 4 °C 24 h Clean and clear, single particles 13342 508 0 0.2 mg/ml PS80 0 h Clean and clear 2733 308 17 4 °C 24 h Clean and clear 4050 283 17 0.4 mg/ml PS80 0 h Clean and clear 1367 175 17 4 °C 24 h Clean and clear 3267 433 8 0.6 mg/ml PS80 0 h Clean and clear 2933 267 0 4 °C 24 h Clean and clear 4167 192 0

Examples 2-4. Screening of sugar types

Different samples were prepared containing 80 mg/mL sucrose, trehalose, and mannitol. Each sample also contained 10 mM succinic acid-sodium succinate (pH 5.0), 80 mg/mL sucrose, and 20 mg/mL (naked antibody protein concentration) ADC-32. Samples were filtered to remove bacteria and particles. Each preparation was filtered and the container was filled, stoppered, and capped. Samples were subjected to high temperature (40 °C) stability testing, -35 °C/4 °C freeze/thaw cycle testing, and appearance, SEC, and recovered CE testing.

The results are shown in Table 9. Under freeze/thaw conditions, sucrose was superior to trehalose and mannitol in appearance. Sucrose and trehalose were superior to mannitol in SEC results. At 40 °C, sucrose was slightly superior to trehalose in reducing CE results.

Table 9. Results of screening of types of saccharides Types of saccharides Terms and Conditions Appearance SEC% Reduced CE% 80 mg/ml sucrose D0 Clean and clear 99.23 97.44 5 freeze cycles/
defrosting Clean and clear 98.88 97.54 40 °C D17 Clean and clear 95.05 98.04 80 mg/ml trehalose D0 Clean and clear 99.23 97.33 5 freeze cycles/
defrosting A small amount of fine particles 98.83 98.36 40 °C D17 Clean and clear 95.38 96.50 80 mg/ml mannitol D0 Clean and clear 99.20 97.55 5 freeze cycles/
defrosting Individual small particles 90.24 96.72 40 °C D17 Clean and clear 95.29 97.28

Examples 2-5. Screening of pH, antibody-drug conjugate concentrations and polysorbates

Containing 10 mM succinic acid-sodium succinate as a buffer and 80 mg/mL sucrose as a stabilizer, the formulas were developed regarding pH, ADC-32 protein concentration (based on naked antibody) and polysorbate concentration as shown in Table 10. High temperature (40 °C) stability study, light study (4 °C, 4500 lx) and -35 °C/4 °C freeze/thaw cycle study were conducted, and the results were statistically analyzed using the least squares method with SEC and reduced CE as the evaluation indices.

The results are shown in Table 11 and Fig. 8. It is shown that the formulations of the present invention are highly dependent on pH. Under the light, shaking and 40°C conditions, a greater decrease in SEC was observed with increasing pH; similarly, under the light, with increasing pH, the reduced CE increased and a greater decrease in the monomer peak was observed, suggesting that the formulations of the present invention should have low pH values to have improved stability. Under the shaking condition, a decreasing trend in SEC was observed with increasing PS80 concentration. No difference in the effect of PS80 on SEC and decrease in CE was observed under other conditions. A greater decrease in the monomer peak SEC at 40°C was observed with increasing ADC-32 protein concentration. No difference in the effect of ADC-32 protein concentration on SEC and decrease in CE was observed under other conditions.

Table 10. Designs of formulas for screening experiments Formula No. pH PS80
(10 ^-4 g/ml) ADC-32 protein concentration (mg/mL, based on naked antibody) 1 5.0 1.7 10 2 5.2 1 30 3 5.5 3 10 4 4.8 2 30 5 4.8 1 20 6 5.5 1 10 7 5.5 2 30 8 4.8 3 16 9 5.5 2 20 10 5.2 3 30 11 5.2 2 20 12 5.2 2 20

Table 11. Formula screening results No. Terms and Conditions Appearance SEC% ΔSEC% Reduced CE% Δ Reduced CE% 1 D0 Clean and clear 99.2 0,0 99.0 0,0 Lighting D10 Clean and transparent, yellowish 88.9 10.3 91.5 7.5 Shaking D15 Clean and clear 97.8 1.4 99.0 0,0 40 °C D15 Clean and clear 96.0 3.2 97.9 1,1 2 D0 Clean and clear 98.9 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 79.4 19.5 90.5 8.2 Shaking D15 Clean and clear 96.3 2.6 99.1 -0.4 40 °C D15 Clean and clear 94.7 4.2 97.9 0.7 3 D0 Clean and clear 98.9 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 77.2 21.7 87.2 11.5 Shaking D15 Clean and clear 96.9 2.0 97.5 1,2 40 °C D15 Clean and clear 95.1 3.8 98.2 0.5 4 D0 Clean and clear 99.0 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 86.3 12.7 91.4 7.3 Shaking D15 Clean and clear 97.7 1.3 98.8 -0.1 40 °C D15 Clean and clear 95.3 3.7 98.1 0.6 5 D0 Clean and clear 99.1 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 90.0 9.1 92.1 6.7 Shaking D15 Clean and clear 97.5 1.6 98.4 0.4 40 °C D15 Clean and clear 95.7 3.4 98.0 0.7 6 D0 Clean and clear 98.9 0,0 98.9 0,0 Lighting D10 Clean and transparent, yellowish 76.2 22.8 86.9 12.0 Shaking D15 Clean and clear 95.0 4.0 98.5 0.4 40 °C D15 Clean and clear 95.2 3.7 98.1 0.8 7 D0 Clean and clear 98.7 0,0 98.3 0,0 Lighting D10 Clean and transparent, yellowish 68.2 30.5 86.8 11.4 Shaking D15 Clean and clear 96.1 2.6 98.9 -0.6 40 °C D15 Clean and clear 94.3 4.4 97.7 0.6 8 D0 Clean and clear 99.2 0,0 99.0 0,0 Lighting D10 Clean and transparent, yellowish 88.3 10.9 91.8 7.2 Shaking D15 Clean and clear 98.1 1.0 98.9 0,1 40 °C D15 A small amount of fine particles 95.8 3.4 98.5 0.5 9 D0 Clean and clear 98.9 0,0 97.6 0,0 Lighting D10 Clean and transparent, yellowish 73.8 25.1 87.1 10.5 Shaking D15 Clean and clear 96.3 2.6 97.7 -0.1 40 °C D15 Clean and clear 94.7 4.1 97.1 0.5 10 D0 Clean and clear 98.9 0,0 98.1 0,0 Lighting D10 Clean and transparent, yellowish 80.9 18.0 90.4 7.7 Shaking D15 Clean and clear 97.3 1.6 99.0 -1.0 40 °C D15 Clean and clear 94.8 4.2 98.5 -0.5 11 D0 Clean and clear 99.1 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 80.5 18.6 88.7 10.0 Shaking D15 Clean and clear 97.2 1.9 98.4 0.3 40 °C D15 Clean and clear 95.4 3.7 98.3 0.3 12 D0 Clean and clear 99.0 0,0 98.7 0,0 Lighting D10 Clean and transparent, yellowish 78.2 20.8 88.4 10.3 Shaking D15 Clean and clear 97.1 1.9 99.1 -0.4 40 °C D15 Clean and clear 95.4 3.6 97.9 0.8

Examples 2-6. Development of a lyophilization process

A 20 mg/mL (protein concentration based on naked antibody) ADC-33 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the parameters of lyophilization process 1 (see Table 12). The sample for lyophilization was a white powder with a flat surface and contained 1.05% water. The lower part of the powder was slightly pressed into the center. The sample was reconstituted with water for injection and the pH value was measured. The results are shown in Table 13. The ionic strength (10 mM or more) was high enough for buffering. The formula included 80 mg/mL sucrose, which can meet the isoosmotic requirement.

Table 12. Parameters of the lyophilization process 1 Parameter of the lyophilization process Temperature setting (°C) Time setting (min) Retention time (h) Degree of vacuum (Pa) Pre-freezing 5 10 1 ND (no data) Pre-freezing -45 50 2 ND Primary drying -25 120 40 10 Secondary drying 25 60 8 1

Table 13. Final results of pH and osmolality determination using the formula pH buffer Osmotic pressure of the initial solution pH of the initial solution pH after restoration 5.00 298 mOsm 5.06 5.06

20 mg/mL (protein concentration based on naked antibody) of ADC-33 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the parameters of lyophilization process 2 (see Table 14). The surface of the powder tablet was flat and free from wrinkles and depressions. The water content was 0.89%. The lyophilization process can meet the quality requirements of the product.

Table 14. Parameters of the lyophilization process 2 Parameter of the lyophilization process Temperature setting (°C) Time setting (min) Retention time (h) Degree of vacuum (Pa) Pre-freezing -5 10 1 ND Pre-freezing -45 40 2 ND Primary drying -20 120 35 10 Secondary drying 25 60 8 1

20 mg/mL (protein concentration based on naked antibody) of ADC-32 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the parameters of lyophilization process 3 (see Table 15). The surface of the powder tablet was flat and had no depressions. The lower edge of the powder tablet was slightly compressed. The water content was 1.17%. The lyophilization process can essentially meet the quality requirements of the product.

Table 15. Parameters of the lyophilization process 3 Parameter of the lyophilization process Temperature setting (°C) Time setting (min) Retention time (h) Degree of vacuum (Pa) Pre-freezing -5 10 1 ND Pre-freezing -45 40 2 ND Primary drying -15 120 30 10 Secondary drying 25 60 9 1

Examples 2-7. Long-term stability test data

A 20 mg/mL (protein concentration based on naked antibody) ADC-32 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered and dispensed, and then the finished batches of the stock solution were formulated according to the lyophilization process parameters 2, subjected to long-term storage at 2-8 °C, reconstituted and then tested. The results are shown in Table 16. Compared with the products at D0, those at 2-8 °C M3 (month 3) conditions showed SEC, reduced CE and free toxin levels within the acceptable ranges (SEC% 93% or more; reduced CE% 95% or more; free toxin level 330 ^ppm or less). SEC decreased slightly by 0.5%, while CE and free toxin levels remained unchanged.

Table 16. Long-term stability test results Batch No. Terms and Conditions SEC% Reduced CE% Free toxin P1927 D0 97.6 99.3 less than 5 million ^-1 2-8 °C M3 (3 months) 97.2 99.2 less than 5 million ^-1 P1929 D0 97.8 99.3 less than 5 million ^-1 2~8 °C M3 97.2 99.2 less than 5 million ^-1

Note: In the table, "M" means month; for example, "M3" means 3 months.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI PHARMACEUTICALS CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> PHARMACEUTICAL COMPOSITION CONTAINING A CONJUGATE

ANTIBODIES AND DRUG AND ITS APPLICATION

<130> 721021CPCT

<150>CN202010219601.7

<151> 2020-03-25

<150>CN202110287012.7

<151> 2021-03-17

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Trastuzumab light chain

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 2

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Trastuzumab heavy chain

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Lys

450

<210> 3

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Pertuzumab light chain

<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Pertuzumab heavy chain

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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 Glu Glu Met 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

<210> 5

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain 1F9DS antibody B7H3

<400> 5

Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met

35 40 45

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

50 55 60

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

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg

85 90 95

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

100 105 110

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

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

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

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys

210 215

<210> 6

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CHAIN

<223> Heavy chain 1F9DS antibody B7H3

<400> 6

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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 (23)

1. A pharmaceutical composition for the treatment of cancer associated with HER2 expression, comprising a conjugate of an antibody and a drug of the following structure: where n is a decimal or integer number from 1 to 10; wherein the pharmaceutical composition contains: (a) an antibody-drug conjugate at a concentration of 10 mg/mL to 30 mg/mL, (b) polysorbate 80 at a concentration of 0.05 mg/mL to 0.5 mg/mL, (c) sucrose at a concentration of 60 mg/mL to 90 mg/mL, and (d) succinic acid-sodium succinate buffer at a concentration of 5 mM to 20 mM; the pharmaceutical composition has a pH from 4.5 to 5.2. 2. A pharmaceutical composition according to claim 1, which has a pH from 4.8 to 5.2. 3. A pharmaceutical composition according to claim 1 or 2, which has a pH from 5.0 to 5.1. 4. A pharmaceutical composition according to any one of claims 1-3, wherein polysorbate 80 is present at a concentration of 0.2 mg/ml. 5. A pharmaceutical composition according to any one of claims 1-4, wherein sucrose is present at a concentration of 80 mg/ml. 6. A pharmaceutical composition according to any one of claims 1-5, wherein the antibody-drug conjugate is present at a concentration of 20 mg/ml to 22 mg/ml. 7. A pharmaceutical composition according to any one of claims 1-6, wherein the succinic acid-sodium succinate buffer is present at a concentration of 10 mM. 8. A pharmaceutical composition according to any one of paragraphs 1-7, containing the following components: (a) an antibody-drug conjugate at a concentration of 20 mg/mL to 22 mg/mL, (b) polysorbate 80 at a concentration of 0.2 mg/mL, (c) sucrose at a concentration of 80 mg/mL, and (d) a succinic acid-sodium succinate buffer at a concentration of 10 mM; wherein the composition has a pH of 5.0-5.1. 9. A pharmaceutical composition according to any one of claims 1-8, wherein the antibody conjugate has the following structure: where n is a decimal or integer number from 3 to 8. 10. A pharmaceutical composition for the treatment of cancer associated with HER2 expression, comprising an antibody-drug conjugate having the following structure: where n is a decimal or integer number from 3 to 8; the pharmaceutical composition comprises (a) a conjugate of an antibody and a drug at a concentration of 20 mg/ml to 22 mg/ml, (b) polysorbate 80 at a concentration of 0.2 mg/ml, (c) sucrose at a concentration of 80 mg/ml and (d) a succinic acid-sodium succinate buffer at a concentration of 10 mM; wherein the pharmaceutical composition has a pH of 5.0-5.1. 11. A method for producing a lyophilized preparation containing a conjugate of an antibody and a drug, comprising a step of lyophilizing the pharmaceutical composition according to any one of claims 1-10. 12. A method for treating a cancer associated with HER2 expression, comprising administering to a patient an effective amount of a pharmaceutical composition according to any one of claims 1-10, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland carcinoma, esophageal cancer, melanoma, neuroglioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.