HK40045552B - Ligand-drug conjugate of exatecan analogue, preparation method therefor and application thereof - Google Patents

Description

Ligand-drug conjugates of ixenostatin analogues, their preparation methods and applications

Technical Field

This disclosure relates to a novel class of ligand-drug conjugates of ixotecan (or ixatecan) analogs. Specifically, this disclosure relates to an antibody-drug conjugate of an ixotecan analog containing structural unit Y, a method for preparing the conjugate, a pharmaceutical composition comprising the conjugate, and the use of the conjugate or pharmaceutical composition.

Background Technology

Chemotherapy remains one of the most important anti-cancer methods, along with surgery, radiotherapy, and targeted therapy. Although there are many types of highly effective cytotoxic drugs, the small difference between tumor cells and normal cells limits the widespread clinical application of these anti-tumor compounds due to their toxic side effects. Anti-tumor monoclonal antibodies, with their specificity to tumor cell surface antigens, have become frontline drugs in anti-tumor therapy; however, when used alone as an anti-tumor drug, the efficacy is often unsatisfactory.

Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxins, while avoiding the drawbacks of low efficacy of the former and excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

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

In August 2011, Adcetris (brentuximab vedotin, Seattle Genetics) received fast-track approval from the US FDA for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). It is a novel targeted ADC drug that allows the drug to directly act on the target CD30 on lymphoma cells, causing endocytosis and inducing apoptosis of tumor cells.

Mylotarg and Adcetris are both targeted therapies for hematologic malignancies, which have relatively simpler tissue structures compared to solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) received FDA approval in the United States for the treatment of HER2-positive patients with advanced or metastatic breast cancer who are resistant to trastuzumab (trade name: Herceptin) and paclitaxel (WO2005037992; US8088387). Kadcyla was the first ADC drug approved by the FDA for the treatment of solid tumors.

Several classes of small molecules with cytotoxicity are used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Reports on the application of camptothecin derivative ethanotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) include WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of more effective ADC drugs is still needed.

Summary of the Invention

To improve the conjugation effect of ligands, particularly antibodies and drugs, this disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvation thereof, wherein the ligand-drug conjugate comprises the structure shown in formula (-D):

in:

Y is selected from -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)- or -S-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^Rb together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

R ¹ is selected from halogens, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

R ² is selected from hydrogen atom, halogen, haloalkyl, deuteralkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

Alternatively, ^Ra and ^R2 together with the carbon atom they are attached to form cycloalkyl or heterocyclic groups;

In this context, the wavy line in -D represents a hydrogen atom, or is covalently linked to a linker unit or to an antibody that binds to the antigen expressed by the target cell;

m is an integer from 0 to 4.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is a compound having the general formula ( _-D1 ) or a ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound:

in:

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form _C3-6 cycloalkyl groups;

The wavy line in Formula- _D1 represents a hydrogen atom, or a linker unit or an antibody that binds to the antigen expressed by the target cell;

m is 0 or 1.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is a ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (Pc-L-Y-Dr):

in:

Y is selected from -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)- or -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^Rb together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

R ¹ is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

R ² is selected from hydrogen atom, halogen, haloalkyl, deuteralkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

Alternatively, ^Ra and ^R2 together with the carbon atom attached to them can form cycloalkyl or heterocyclic groups;

m is an integer from 0 to 4;

n can be 1 to 10, and can be an integer or a decimal;

Pc represents the ligand; L represents the connector unit.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is described.

Where -Y- is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens or alkyl groups;

^R1 is a _C3-6 cycloalkyl or _C3-6 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

m is 0 or 1.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is provided, wherein the structural unit -Y- is -O-( _CH2 )m- ^CR1R2 - ^C (O)-;

^R1 is a _C3-6 cycloalkyl or _C3-6 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form _C3-6 cycloalkyl groups;

m is 0 or 1.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is provided, wherein the structural unit -Y- is -O-( _CH2 )m- ^CR1R2 - ^C (O)-;

^R1 is a _C3-6 cycloalkyl or _C3-6 cycloalkyl;

^R2 is a hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

m is 0 or 1.

In some embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is provided, wherein the structural unit -Y- is -O-( _CH2 )m- ^CR1R2 - ^C (O)-;

^R1 is a _C3-6 cycloalkyl or _C3-6 cycloalkyl;

^R2 is a hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

m is 0.

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is wherein the structural unit -Y- is selected from:

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvate is wherein the O end of the -Y- is connected to the connector unit L.

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvate compound is a ligand-drug conjugate or its pharmaceutically acceptable salt or solvate of the general formula (Pc-L-D1):

in:

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form _C3-6 cycloalkyl groups;

m is 0 or 1;

n can be 1 to 10, and can be an integer or a decimal;

Pc represents the ligand; L represents the connector unit.

In another preferred embodiment of the invention, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to the invention, wherein n is 2 to 8, which can be an integer or a decimal; preferably 3 to 8, which can be an integer or a decimal.

In another preferred embodiment of the invention, the ligand-drug conjugate according to the invention or its pharmaceutically acceptable salt or solvate, wherein the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is selected from -(succinimide-3-yl-N)-WC(O)-, _-CH2 -C(O) ^-NR3- WC(O)-, or -C(O)-WC(O)-, wherein W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl, or a straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O, or S, wherein the _C1-8 alkyl, cycloalkyl, and straight-chain heteroalkyl are each optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, and cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 _{( CH2CH2O} ) _p1CH2C (O)-, ^-S ( _CH2 ) ^p1C (O)- or chemical bonds, wherein ^p1 is an integer from ¹ _{to 20} ; preferably a chemical bond;

^L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids may optionally be further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl.

^L4 is selected from ^-NR5 ( ^CR6R7 ) _t- , -C(O) ^NR5 , -C(O) ^NR5 ( _CH2 ⁾ _t- or chemical bonds, where t is an integer ^from 1 to 6; preferably ^-NR5 ( ^CR6R7 )t-;

^R3 , ^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound, wherein the linker unit ^L1 is selected from -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)-, -(succinimide-3-yl-N)-CH2 _- cyclohexyl-C(O)-, -(succinimide-3- _yl -N)-( _CH2CH2O ) ^s2- _CH2CH2 - _C (O)-, _-CH2 -C(O) ^-NR3- ( _CH2 ) ^s3 -C(O)- or -C(O)-( _CH2 ) ^s4C (O)-, wherein ^s1 is an integer from 2 to 8, ^s2 is an integer from 1 to 3, ^s3 is an integer from 1 to 8, and ^s4 is an integer from 1 to 8; ^s1 is preferably 5.

In other embodiments of this disclosure, the ligand-drug conjugate or ^its pharmaceutically acceptable salt or solvent compound is wherein the linker unit ^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or a chemical bond, _and ^p1 is an integer from 6 to 12.

In other embodiments of this disclosure, the provided ligand-drug conjugate or ^its pharmaceutically acceptable salt or solvent compound is provided, wherein ^L4 is selected from ^-NR5 ( ^CR6R7 )t-, ^R5 is selected from hydrogen atom or alkyl, ^R6 and ^R7 are the same or different and are each independently hydrogen atom or alkyl, t is 1 or 2, preferably 2; ^L4 is preferably from ^{-NR5CR6R7- ; L4} is more preferably _from ^-NHCH2- .

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is provided, wherein the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is an integer from 2 to 8 for ^s1 ;

^L2 is a chemical bond;

^L3 is a tetrapeptide residue;

^L4 is ^-NR5 ( ^CR6R7 )t-, where ^R5 is selected from hydrogen atoms or alkyl groups, ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group, ^and t is 1 or 2.

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound is provided, wherein the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is -(succinimide-3-yl-N) _-CH2 -cyclohexyl-C(O)-;

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

^L3 is a tetrapeptide residue;

^L4 is ^-NR5 ( ^CR6R7 )t-, where ^R5 is selected from hydrogen atoms or alkyl groups, ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group, ^and t is 1 or 2.

In other embodiments of this disclosure, the provided ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound, wherein the peptide residue of ^L3 is an amino acid residue formed from one, two or more amino acids selected from 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 phenylalanine and glycine; more preferably a tetrapeptide residue; and most preferably a GGFG (glycine-glycine-phenylalanine-glycine) tetrapeptide residue.

In other embodiments of this disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the connector unit-L- has its ^L1 end connected to the ligand and its ^L4 end connected to Y.

In other embodiments of this disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein -L-Y- is:

^L1 is selected from -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)- or -(succinimide-3-yl-N)-CH2 _- cyclohexyl-C(O)-;

^L2 is ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or _a chemical bond, ^{where p1} is an integer from 6 to 12;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8; preferably 5;

m is an integer from 0 to 4.

In other embodiments of this disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein -L-Y- is:

Preferred options are:

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

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

m is an integer from 0 to 4.

In other embodiments of this disclosure, the ligand-drug conjugate provided by the general formula (Pc-L-Y-Dr) or its pharmaceutically acceptable salt or solvent compound, wherein -L-Y- is

^L2 is a chemical bond;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8; preferably 5;

m is an integer from 0 to 4.

Another aspect of this disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises the structure shown in formula (-L-Y-):

It can be used to obtain ligand-drug conjugates formed by linking drugs and ligands via linker fragments;

in:

^L1 is selected from -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)- or -(succinimide-3-yl-N)-CH2 _- cyclohexyl-C(O)-;

^L2 is ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or _a chemical bond, ^{where p1} is an integer from 1 to 20;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

^R5 , ^R6 , or ^R7 may be the same or different, and each may be an independent hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer from 0 to 4.

Another aspect of this disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises the structure shown in formula (-L-Y-):

in:

^L2 is a chemical bond;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-6 cycloalkyl or _C3-6 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer from 0 to 4.

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (Pc- _LY -Dr) is provided, which is the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (Pc-L a-Y-Dr):

in:

W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl, or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O, or S, wherein each of the _C1-8 alkyl, cycloalkyl, and straight-chain heteroalkyl is optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, and cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ⁾ _p1CH2C (O)-, -S _{( CH2} ) ^p1C (O)- or chemical bonds, ^{where p1} is an integer from ₁ to ₂₀ ;

^L3 is a peptide residue consisting of 2 to 7 amino acids, which may be substituted or unsubstituted. When substituted, the substituent may be substituted at any usable linker site. The substituent is one or more independently selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuteralkyl groups, alkoxy groups, and cycloalkyl groups.

R ¹ is selected from halogens, cycloalkylalkyl groups, deuterated alkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; preferably cycloalkylalkyl groups or cycloalkyl groups; more preferably _C3-6 cycloalkylalkyl groups or _C3-6 cycloalkyl groups;

^R2 is selected from hydrogen atoms, halogens, haloalkyls, deuterated alkyls, cycloalkyls, heterocyclic groups, aryl groups, or heteroaryl groups; preferably hydrogen atoms; or, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyls or heterocyclic groups;

^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

m is an integer from 0 to 4;

n is a non-zero integer or decimal between 0 and 10, preferably an integer or decimal between 1 and 10;

Pc is the ligand.

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (Pc-La-Y-Dr) is provided, which is the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (Pc- _Lb -Y-Dr):

in:

^s1 is an integer from 2 to 8; preferably 5;

Pc, ^R1 , ^R2 , ^R5 to ^R7 , m and n are defined as in the general formula (Pc-La-Y-Dr).

The linker unit -L-Y - of the ligand-drug conjugate disclosed herein includes, but is not limited to:

The ligand-drug conjugates represented by the general formula (Pc-L-Y-Dr) disclosed herein include, but are not limited to:

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

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvate is wherein the Pc is an antibody or its antigen-binding fragment, and the antibody is selected from chimeric antibodies, humanized antibodies or fully human antibodies; preferably monoclonal antibodies.

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody or its antigen-binding fragment.

In other embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab, or its antigen-binding fragment.

The ligand-drug conjugates represented by the general formula (Pc-L-Y-Dr) disclosed herein include, but are not limited to, the following structural formulas:

Wherein, n is a non-zero integer or decimal from 0 to 10, preferably an integer or decimal between 1 and 10; more preferably 2 to 8, which can be an integer or a decimal; most preferably 3 to 8, which can be an integer or a decimal.

Another aspect of this disclosure provides a compound of general formula (D) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

in:

Y is selected from -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)- or -S-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^Rb together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

R ¹ is selected from halogens, cycloalkylalkyl groups, deuterated alkyl groups, cycloalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

R ² is selected from hydrogen atom, halogen, haloalkyl, deuteralkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

Alternatively, ^Ra and ^R2 together with the carbon atom attached to them form cycloalkyl or heterocyclic groups;

m is an integer from 0 to 4.

In another aspect of this disclosure, a preferred embodiment is provided, in the form of a compound of general formula (D), or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which is a compound of general formula ( _D1 ) or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

Wherein: ^R1 is a _C3-6 cycloalkyl or a _C3-6 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, or _C3-6 cycloalkyl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form _C3-6 cycloalkyl groups;

m is 0 or 1.

The compounds represented by general formula (D) described in this disclosure include, but are not limited to:

Or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof.

In another preferred embodiment of this disclosure, a compound of the general formula ( _La -Y-Dr) or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, is provided:

in

W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl, or a straight-chain heteroalkyl group of 1 to 8 atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O, or S, wherein each of the _C1-8 alkyl, cycloalkyl, and straight-chain heteroalkyl groups is independently and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, and cycloalkyl.

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ⁾ _p1CH2C (O)-, -S _{( CH2} ) ^p1C (O)- or chemical bonds, where ^{p1 is} an integer from ₁ to ₂₀ ;

^L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. When substituted, the substituents can be substituted at any usable linker site. The substituents are one or more independently selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups.

R ¹ is selected from halogens, cycloalkylalkyl groups, deuterated alkyl groups, cycloalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

^R2 is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; or, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

m is an integer from 0 to 4.

In another preferred embodiment of this disclosure, a compound of the general formula (L _{_a} -Y-Dr) or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically usable salt thereof, is provided.

^R1 , ^R2 , ^R5 to ^R7 , ^s1 and m are defined as in the general formula ( _La -Y-Dr).

The compounds represented by the general formula ( _La -Y-Dr) described in this disclosure include, but are not limited to:

Or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof.

Another aspect of this disclosure provides a method for preparing a compound of general formula ( _D1 ) or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically usable salts thereof, comprising the following steps:

The condensation reaction of general formula ( _Y1 ) and general formula (Dr) yields the compound shown in general formula ( _D1 ).

Where ^R1 , ^R2 and m are as defined in general formula ( _D1 ).

Another aspect of this disclosure provides a method for preparing a compound of the general formula ( _Lb -Y-Dr) or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, comprising the following steps:

The condensation reaction of general formulas (IA) and (IB) yields a compound represented by the general formula ( _Lb -Y-Dr).

Where ^R1 , ^R2 , ^R5 to ^R7 , ^s1 and m are as defined in the general formula ( _Lb -Y-Dr).

Another aspect of this disclosure provides a method for preparing a compound of the general formula ( _Lb -Y-Dr) or its tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, comprising the following steps:

The compounds of general formula (IA) and general formula (IB) undergo a condensation reaction to give the compound represented by general formula ( _Lb -Y-Dr).

Where ^R1 , ^R2 , ^R5 to ^R7 , ^s1 and m are as defined in the general formula ( _Lb -Y-Dr).

Another aspect of this disclosure provides a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in the general formula (Pc- _La -Y-Dr), comprising the following steps:

After reduction, Pc is coupled with a general formula ( _La -Y-Dr) to obtain a general formula (Pc- _La -Y-Dr); the preferred reducing agent is TCEP.

in:

Pc is the ligand;

W, ^L2 , ^L3 , ^R1 , ^R2 , ^R5 ~ ^R7 , m and n are defined as in the general formula (Pc-L _a -Y-Dr).

Another aspect of this disclosure further relates to a pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate or compound as described in this disclosure, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

Another aspect of this disclosure further relates to a pharmaceutical composition comprising a compound represented by general formula (D) according to this disclosure, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

Another aspect of this disclosure further relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising a ligand and a drug attached to the ligand, wherein the drug is selected from compounds of general formula (D) of this disclosure, compounds of general formula ( _La -Y-Dr), or tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, preferably the drug being attached to the ligand via a linker, preferably the ligand being a monoclonal antibody.

Another aspect of this disclosure further relates to a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising the step of linking a compound of general formula (D), a compound of general formula ( _La -Y-Dr), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, to a ligand, preferably via a linker, preferably a monoclonal antibody.

Another aspect of this disclosure further relates to the ligand-drug conjugates or compounds described herein, or pharmaceutically acceptable salts or solvates thereof, or pharmaceutical compositions thereof, used as pharmaceuticals.

Another aspect of this disclosure further relates to the use of the ligand-drug conjugates or compounds described herein, or pharmaceutically acceptable salts or solvates thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for the treatment or prevention of tumors; preferably, the tumors described herein are cancers associated with HER2, HER3, or EGFR expression.

Another aspect of this disclosure further relates to the use of the ligand-drug conjugates or compounds described herein, or pharmaceutically acceptable salts or solvates thereof, or pharmaceutical compositions, in the preparation of medicaments for treating and/or preventing cancer, preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, prostate cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

Another aspect of this disclosure further relates to a method for treating and/or preventing tumors, the method comprising administering to a patient in need a therapeutically effective dose of the ligand-drug conjugate or compound described in this disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising thereof; preferably wherein the tumor is a cancer associated with HER2, HER3, or EGFR expression.

Another aspect of this disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a patient in need a therapeutically effective dose of the ligand-drug conjugate or compound described in this disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising thereof; wherein the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

The active compound can be formulated into a form suitable for administration via any appropriate route, preferably in a unit dose manner or in a manner that allows the patient to self-administer a single dose. The unit dose of the compound or composition of the present invention can be expressed as a tablet, capsule, sachet, bottled liquid, powder, granule, lozenge, suppository, regenerated powder, or liquid formulation.

The dosage of compounds or compositions used in the treatment methods of this invention will generally vary depending on the severity of the disease, the patient's weight, and the relative efficacy of the compounds. However, as a general guideline, a suitable unit dose may be 0.1 to 1000 mg.

In addition to the active compound, the pharmaceutical compositions of the present invention may contain one or more excipients selected from the following components: fillers (diluents), binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

Pharmaceutical compositions containing active ingredients can be in forms suitable for oral administration, such as tablets, sugar lozenges, tablets, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions. Such compositions may contain binders, fillers, lubricants, disintegrants, or pharmaceutically acceptable wetting agents, and may also contain one or more ingredients selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide an appealing and palatable pharmaceutical formulation.

Aqueous suspensions contain active substances and excipients suitable for preparing aqueous suspensions for mixing. Aqueous suspensions may also contain one or more preservatives, one or more colorants, one or more flavoring agents, and one or more sweeteners.

Oil suspensions are prepared by suspending the active ingredient in vegetable oil. Oil suspensions may contain thickeners. Sweeteners and flavoring agents mentioned above may be added to provide a palatable formulation.

The pharmaceutical composition may also provide the active ingredient as a dispersible powder or granules for preparing an aqueous suspension, by adding one or more of a water-mixing dispersant, wetting agent, suspending agent, or preservative. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid.

The pharmaceutical compositions disclosed herein may also be in the form of oil-in-water emulsions.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or media that can be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the patient's bloodstream by local large-volume injection. Alternatively, it is preferable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the compound disclosed herein. To maintain such a constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous infusion pump.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a parenteral-acceptable, non-toxic diluent or solvent. Furthermore, sterile fixative oils may be conveniently used as a solvent or suspension medium.

The disclosed compounds can be administered in suppository form for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable, non-irritating excipient that is solid at normal temperatures but liquid in the rectum, and thus dissolves in the rectum to release the drug. Such substances include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycol of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health status, the patient's behavior, the patient's diet, the timing of administration, the route of administration, the rate of excretion, and the combination of drugs; in addition, the optimal treatment mode, such as the treatment pattern, the daily dosage of the general formula compound (I), or the type of pharmaceutically acceptable salt, can be validated based on conventional treatment protocols.

Attached Figure Description

Figure 1A: Plasma stability test results of ADC-19 disclosed herein.

Figure 1B: Plasma stability test results of ADC-18 disclosed herein.

Figure 1C: Plasma stability test results of ADC-20 disclosed herein.

Figure 2: Evaluation of the efficacy of ADC-21 and ADC-24 in JIMT-1 tumor-bearing mice.

Figure 3: Evaluation of the efficacy of the ADC disclosed in this paper on nude mice with human breast cancer cell SK-BR-3 xenograft tumors.

Figure 4: Plasma stability test results of ADC-25 disclosed herein.

Figure 5: The efficacy of the ADC disclosed in this study on human astrocytoblastoma U87MG xenografts in nude mice.

Figure 6: The efficacy of the ADC disclosed herein on Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

Figure 7: The efficacy of the ADC disclosed herein on human glioma U87MG xenografts in nude mice.

Detailed Implementation

Detailed description of the invention

Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

When a trade name is used in this disclosure, the applicant intends to include formulations of products under that trade name, generic drugs of products under that trade name, and active pharmaceutical ingredients.

Unless otherwise stated, the terms used in the specification and claims have the following meanings.

The term "ligand" is a macromolecular compound that recognizes and binds to antigens or receptors associated with target cells. The role of ligands is to deliver drugs to the target cell population that has bound to them. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In embodiments of this disclosure, the ligand is designated as Pc. The ligand can form a linker bond with a linker unit via heteroatoms on the ligand, preferably an antibody or its antigen-binding fragment, selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; preferably monoclonal antibodies.

The term "drug" refers to a cytotoxic drug, denoted as Dr, which is a chemical molecule that strongly disrupts the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. This term also includes toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals; radioactive isotopes (e.g., radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , and Lu); toxic drugs; chemotherapeutic agents; antibiotics; and ribolysins, preferably toxic drugs.

The term "connector unit,""linkingsegment," or "linking unit" refers to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It may also be connected to other connectors before being linked to the drug. The preferred embodiment of this disclosure is represented by L and ^L1 to ^L4 , wherein ^L1 is connected to the ligand, and ^L4 is connected to the structural unit Y and then to the drug (Dr).

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

The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In this disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker unit.

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

The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain. The antibodies described in this disclosure are preferably specific antibodies against cell surface antigens on target cells. Non-limiting examples include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, and anti-Integr antibody. The antibody is selected from one or more of the following: anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody; preferably trastuzumab (trade name Herceptin), pertuzumab (also known as 2C4, trade name Perjeta), nimotuzumab (trade name Taixinsheng), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, and glematumamab.

The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk/vbase ) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Further literature describing methods that can be used to participate in humanization includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter et al. [Jones et al., Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. Fragments of full-length antibodies have been shown to be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally connected by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH₂ _- VL-linker-VH-COOH or NH₂ _- VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using 1–4 repeating variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain.

The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

The term "epitope" or "antigenic determinant" refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms "specific binding,""selectivebinding,""selectivebinding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen. Typically, antibodies bind with an affinity (KD) of approximately less than ^10⁻⁷ M, such as approximately less than ^10⁻⁸ M, ^10⁻⁹ M, or ^10⁻¹⁰ M or less.

The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

The term "peptide" refers to a compound fragment that lies between amino acids and proteins. It is composed of two or more amino acid molecules linked together by peptide bonds. It is a structural and functional fragment of proteins, such as hormones and enzymes, which are essentially peptides.

The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O. It can be classified into monosaccharides, disaccharides, and polysaccharides.

The term "fluorescent probe" refers to a class of fluorescent molecules that exhibit characteristic fluorescence in the ultraviolet-visible-near-infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, and polarization, etc.) can be sensitively altered by changes in the properties of their environment, such as polarity, refractive index, and viscosity. These fluorescent probes interact non-covalently with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, causing changes in one or more fluorescence properties. They can be used to study the properties and behavior of macromolecules.

The term "toxic drug" refers to substances that inhibit or prevent cell function and/or cause cell death or damage. This includes toxins and other compounds that can be used in cancer treatment.

The term "toxin" refers to any substance that can have a harmful effect on cell growth or proliferation. It can be a small molecule toxin from bacteria, fungi, plants, or animals, and its derivatives, including camptothecin derivatives such as ixatecan, maytansine alkaloids and their derivatives (CN101573384) such as DM1, DM3, and DM4, auristatin F(AF) and its derivatives such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria toxin, exotoxins, ricin A chain, abrin A chain, modeccin, α-sarcin, and aleutites fordii. Toxins, carnation (dianthin) toxins, American pokeweed (PAPI, PAPII and PAP-S) toxins, bitter melon (Momordica charantia) inhibitors, jatropha curcin, crotin, soapwort (Sapaonaria officinalis) inhibitors, white tree toxins (gelonin), mitogellin, restrictedocin, phenomycin, enomycin, and trichothecenes.

The term "chemotherapy drug" refers to chemical compounds that can be used to treat tumors. This definition also includes anti-hormonal agents that modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They themselves can be hormones. Examples of chemotherapy drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN ^™ ); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benaodopa, carboquone, meturedopa, and uredopa; aziridines and methylamelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolamine; and nitrogen mustard. Mustards include chlorambucil, naphthylmustine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and oxynitrogen hydrochloride; melphalan, novobichin, cholesterol phenylacetic acid mustard, prednimustine, trofosfamide, and uracil mustard; and nitrosureas include carmustine, chlorozotocin, and formostin. e) Lomustine, Nimustine, Ranimustine; antibiotics such as aclarubicin, actinomycin, authramycin, diazoserine, bleomycin, cactinomycin C, calicheamicin, carabicin, chromomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin Rubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, 5 - Fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroxate, trimetrexate; pterin analogs such as fludarabine, 6-mercaptopterin, thiophene, thioguanopterin; pyrimidine analogs such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxitluridine, enocitabine, fluorouridine, 5-FU; androgens such as calusterone, dromostanolong. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; acegluconeol; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntrene; edatraxate; defofamine; colchicine; diaziquone; elfomithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pintostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazine; procarbazine; razoxane ; sizofiran; spirogermanium; Alternaria alternata ketone acid; triaminequinone; 2,2',2"-trichlorrotriethylamine;urethan;vinblastamide;dacarbazine; mannitol mustard; mitobronitol; dibromo-euonymol; piperobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa; taxanes, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Rhone-Poulenc) Rorer, Antony, France); Chlorobutyrate; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunorubicin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Esperamicins; Capecitabine ; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing substances. This definition also includes anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogenic agents including tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; and anti-androgenic agents such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing substances.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents are preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

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

The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene ( _-CH2- ), 1,1-ethylene (-CH( _{CH3 )} -), 1,2-ethylene ( _{-CH2CH2 )} -, 1,1-propylene (-CH( _CH2CH3 )-), 1,2-propylene ( _{-CH2CH ( CH3} )-), 1,3-propylene ( _{-CH2CH2CH2- )} , _1,4 -butylene _{( -CH2CH2CH2CH2-} ) _, and _{1,5 - butylene ( -CH2CH2CH2CH2CH2-} ). The alkylene group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

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

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

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

The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 cyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 cyclic, more preferably 7 to 10 cyclic. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups according to the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospirocyclic group. Non-limiting examples of spiroheterocyclic groups include:

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

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

The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

wait.

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

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrrole, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenemethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be replaced by 1-3 substituents selected from halogens, alkoxy groups, or nitro groups. The amino protecting group is preferably 9-fluorenemethoxycarbonyl.

The term "cycloalkylalkyl" refers to an alkyl group that is substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above.

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

The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

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

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

The term "amino" refers to _-NH2 .

The term "nitro" refers to _-NO2 .

The term "amide group" refers to -C(O)N (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuterontetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

"Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically/pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically/pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the ligand-drug conjugate of this disclosure, or a salt of the compounds described in this disclosure, which is safe and effective in mammals and has the intended biological activity. The antibody-antibody-drug conjugate of this disclosure contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

The terms "solvent" or "solvent compound" refer to the ligand-drug conjugate of this disclosure that forms a pharmaceutically usable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

The term "drug loading" refers to the average number of cytotoxic drugs loaded onto each ligand in the molecule of formula (I), and can also be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 0-12, preferably 1-10, cytotoxic drugs (D) linked to each ligand (Pc). In embodiments of the present invention, the drug loading is expressed as n, which can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as exemplarily. The average number of drugs per ADC molecule after the coupling reaction can be identified using conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, and HPLC characterization.

In one embodiment of this disclosure, the cytotoxic drug is coupled to the ε-amino group of the N-terminal amino group and/or lysine residue of the ligand via a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

(1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

(2) Control the reaction time and temperature.

(3) Choose different reaction reagents.

For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

The term "excipient" refers to any additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API). Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation. Examples of absorbents include starch, lactose, inorganic salts of calcium, and microcrystalline cellulose.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Water, Ringer's solution, and isotonic sodium chloride solution may be used as acceptable solvents and media. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the patient's bloodstream by local large-volume injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound disclosed herein. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous infusion pump.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

This disclosure relates to a class of cleavable linker arms of a specific structure and an active ingredient of a specific structure, and antibody-drug conjugates (ADCs) composed of the linker arms, the active ingredient, and an antibody. Such ADCs are complexes formed by linking a toxic substance to an antibody via a spacer. The antibody-drug conjugate (ADC) degrades in vivo to release the active molecule, thereby exerting an antitumor effect.

The synthesis method disclosed herein

To achieve the synthetic objective of this disclosure, the following synthetic technique is adopted:

Option 1:

The preparation method of the compound represented by general formula (D1) of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

Compounds of general formula (Y1) and (Dr) react, optionally under basic conditions, in the presence of a condensing agent, to give compound of general formula (D1).

Where ^R1 , ^R2 and m are as defined in general formula (D1).

The reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide. The inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

The condensing agent may be selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N N',N'-Tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazole-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Option 2:

The present disclosure discloses a method for preparing a compound of general formula ( _Lb -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

In the first step, the compound of general formula (IB-1) and eczetidine mesylate (1b) are reacted under alkaline conditions in the presence of a condensing agent to give the compound of general formula (IB-2).

The second step involves removing the protecting group from the compound of general formula (IB-2) to obtain the compound of general formula (IB).

In the third step, compounds of general formula (IA) and general formula (IB) are reacted, optionally under basic conditions, in the presence of a condensing agent, to yield compound of general formula ( _Lb -Y-Dr).

in:

^Rc is an amino protecting group; preferably 9-fluorenylmethoxycarbonyl (Fmoc);

^R1 , ^R2 , ^R5 to ^R7 , ^s1 and m are defined as in the general formula ( _Lb -Y-Dr).

The reagents that provide alkaline conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide. The inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

The condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N, N',N'-Tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazole-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Option 3:

This disclosure describes a method for compounds of the general formula (Pc-L _a -Y-Dr), comprising the following steps:

After reduction of Pc, it reacts with a general formula ( _La -Y-Dr) to obtain a general formula (Pc- _La -Y-Dr); the reducing agent is preferably TCEP, and in particular, it is preferred to reduce the disulfide bonds on the antibody;

in:

Pc is the ligand;

W, ^L2 , ^L3 , ^R1 , ^R2 , ^R5 ~ ^R7 , m and n are defined as in the general formula (Pc-L _a -Y-Dr).

The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

Example

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ), and deuterated methanol ( _CD3OD ). The internal standard was tetramethylsilane (TMS). Chemical shifts are given in units of ^10⁻⁶ (ppm).

MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

UPLC measurements were performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

The UV-HPLC determination was performed using a Thermo nanodrop2000 UV spectrophotometer.

The proliferation inhibition rate and _IC50 value were determined using a PherastarFS microplate reader (BMG GmbH, Germany).

Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC are 0.15mm to 0.2mm in diameter, while those used for TLC separation and purification are 0.4mm to 0.5mm in diameter.

Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the carrier.

The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc., and Darui Chemicals.

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

Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

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

Room temperature is the optimal reaction temperature, with a range of 20℃ to 30℃.

Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g _{of KH2PO4} , 8.56g _{of K2HPO4 ·} 3H2O, 5.85g of NaCl and 1.5g of EDTA and place them in a bottle, make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

Some of the compounds disclosed herein were characterized by Q-TOF LC/MS. The Q-TOF LC/MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm 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]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

Add 1 mL of N,N-dimethylformamide to eczema sulfonate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopropylformic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (3.8 mg, 13.7 μmol) to the reaction solution in sequence. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%).

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

¹ H NMR (400MHz, CDCl ₃ ): δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H),3.40-3. 15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H).

Example 2

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

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

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (4 mg, 7.53 μmol), purge three times with argon, cool to 0-5 °C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared using the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) sequentially to the reaction solution. After the additions are complete, stir the reaction mixture at 0-5 °C for 1 hour. Remove the ice-water bath, heat to 30 °C, and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridgePrep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ), B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

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

Single-configuration compound 2-B (shorter retention time)

UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1 H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.1 3(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 2-A with a single configuration (longer retention time)

UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

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

Example 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]indolazino[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]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (5.0 mg, 9.41 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath, heat to 30 °C, and stir for 8 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (1.5mg, 1.5mg).

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

Single-configuration compounds (shorter retention time)

UPLC analysis: retention time 1.11 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5. 61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H ),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H) ,2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

Single-configuration compounds (longer retention time)

UPLC analysis: retention time 1.19 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1H),4.66-4 .57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

Example 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]indolazino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 4

Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared using the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction mixture at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with 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 (400MHz, CDCl ₃ ): δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10(m,1H),2 .43(s,3H),2.28-2.20(m,2H),2.08-1.84(m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H).

Example 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]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

Add 1 mL of N,N-dimethylformamide to 1b (2.0 mg, 3.76 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared using the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2 mg, 7.24 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 5 (1.0 mg, yield: 50%).

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

¹ H NMR (400MHz, CDCl ₃ ): δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48(m,1H),3 .30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35-2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H).

Example 6

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

Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared according to the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with 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 (400MHz, DMSO-d ₆ ): δ7.85-7.62(m,1H),6.88(br,1H),5.87-5.48(m,2H),5.47-5.33(m,1H),5.31-5.06(m,1H),4.25-3.91(m,2H),3.25(br,1 H),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 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]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclobutane-1-carboxamide 7

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplier's reagent), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, and purify the residue by thin-layer chromatography using developing solvent system B to give title product 7 (2.5 mg, yield: 83.1%).

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

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5. 59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.1 5-3.05(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 8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-

8-(2-)cyclopropane-1-carboxamide

first step

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid benzyl ester 8c

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

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

Step 2

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

Dissolve 8c (50 mg, 0.10 mmol) in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), add palladium on carbon (25 mg, 10%), purge three times with hydrogen, and stir at room temperature for 1 hour. Filter the reaction solution with diatomaceous earth, wash the filter cake with tetrahydrofuran, concentrate the filtrate to give the title product 8d (41 mg, yield: 100%).

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

Step 3

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)2-oxoethyl)carbamate 8e

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

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

Step 4

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

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

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

Step 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. 0.3 mL of N,N-dimethylformamide solution containing 8 g (2.7 mg, 5.80 μmol, prepared using the method disclosed in patent application "EP2907824") of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrolo-1-yl)hexamylamino)acetamido)acetamido)-3-phenylpropionic acid was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol). The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was heated to room temperature and stirred for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).

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

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4 H),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,1 H),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),2.37-2.2 3(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 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

first step

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

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

Step 2

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 9b

Add 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) to the reaction flask, add 4 mL of tetrahydrofuran, purge three times with argon, cool to 0-5 °C in an ice-water bath, add potassium tert-butoxide (109 mg, 0.98 mmol), remove the ice bath, raise to room temperature and stir for 40 minutes, add 10 mL of ice water, and extract with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). Combine the organic phases and concentrate. Dissolve the residue in 4 mL of dioxane, add 2 mL of water, add sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorene methyl chloroformate (126 mg, 0.49 mmol), and stir at room temperature for 2 hours. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 9b (48 mg, yield: 19%).

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

Step 3

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid 9c

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

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

Step 4

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

Add 1b (10 mg, 18.8 μmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add crude product 9c (13 mg, 30.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (16.9 mg, 61.2 μmol), and stir in an ice bath for 40 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by thin-layer chromatography using developing solvent system B to give the title product 9d (19 mg, yield: 73.6%).

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

Step 5

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

9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 9e (17 mg). The product was used directly in the next reaction without purification.

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

Step 6

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (21.2 mg, 44.8 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

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

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

UPLC analysis: retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7 .26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1H),4.54-4 .40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m,3H), 2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

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

UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.2 6-7.13(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 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]indolazino[1,2-b]quinoline-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-pyrrole-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-B

Step 1: Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

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

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

Step 2

1-(9H-fluorene-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 10b

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

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

Step 3

1-(9H-fluorene-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazabutane-11-acid 10c

10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (15 mg, 10%) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to give the crude product 10c (13 mg).

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

Step 4

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-1,1,1-trifluoro-3-oxopropyl-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

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

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

Step 5

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

10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. This process was repeated three times. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 10e (12 mg). The product was used directly in the next reaction without purification.

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

Step 6

N-((2S,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-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-pyrrole-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-B

Crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, 8 g (14 mg, 29.6 μmol) of 0.3 mL of N,N-dimethylformamide solution was added, followed by 15 mg (54.2 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride. The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ) B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2.7mg, 2.6mg).

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

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.18 min, purity: 91% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.31(s,1H), 7.26-7.10(m,4H),6.99(s,1H),6.66(br,1H),6.52(s,1H),5.65-5.54(m,1H),5.41(s,1H),5.37-5.25(m,3H),5.23-5. 13(m,1H),4.81-4.68(m,2H),4.51-4.41(m,1H),3.78-3.45(m,6H),3.21-3.13(m,1H),3.02-2.93(m,1H),2.77-2.63(m ,2H),2.45-2.29(m,3H),2.24-2.05(m,3H),2.04-1.93(m,5H),1.90-1.75(m,2H),1.52-1.38(m,4H),0.90-0.78(m,5H).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.23 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.05(d,1H),8.97-8.88(m,1H),8.35-8.27(m,1H),8.11-8.03(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.34(s,1H), 7.29-7.13(m,4H),6.99(s,1H),6.66(br,1H),6.54(s,1H),5.64-5.55(m,1H),5.43(s,1H),5.36-5.20(m,3H),4.92-4. 85(m,1H),4.82-4.72(m,2H),4.52-4.42(m,1H),3.77-3.48(m,6H),3.21-3.14(m,1H),3.03-2.95(m,1H),2.79-2.65(m ,2H),2.47-2.28(m,3H),2.25-2.05(m,3H),2.05-1.94(m,5H),1.91-1.76(m,2H),1.52-1.37(m,4H),0.92-0.77(m,5H).

Example 11

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11

first step

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclobutane-1-carboxylic acid benzyl ester 11b

1-Hydroxycyclobutane-benzyl carboxylate 11a (167 mg, 0.81 mmol, prepared according to the method disclosed in the literature "Journal of Medicinal Chemistry, 2013, vol. 56, #13, p. 5541-5552") and 8b (150 mg, 0.41 mmol) were added to a reaction flask, followed by the addition of 5 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then potassium tert-butoxide (92 mg, 0.82 mmol) was added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of dioxane, and 0.6 mL of water was added. Sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorene methyl chloroformate (105 mg, 0.41 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (8 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system C to give title product 11b (37 mg, yield: 17.6%).

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

Step 2

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

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

Step 3

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclobutoxy)methyl)amino)2-oxoethyl)carbamate 11d

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

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

Step 4

1-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11e

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

Step 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11

Crude product 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (1.5 mg, 3.17 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was evaporated to dryness using an oil pump to remove DMF. The residue was dissolved in DCM and purified twice by thin-layer chromatography (evolving solvent polarity: DCM/MeOH = 10/1) to obtain the title product 11 (1 mg, yield: 28.8%).

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

¹ H NMR (400MHz, CDCl ₃ ): δ8.70-8.60(m,1H),8.28-8.19(m,1H),8.13-7.91(m,3H),7.79-7.71(d,1H),7.29(s,1H),7.25-7.09(m,4H),6.9 8(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 12

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

first step

12b of 3-cyclopropyl-2-hydroxypropionic acid

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

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

Add 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide to 1b (45 mg, 0.085 mmol), purge three times with argon, add 0.1 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 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) sequentially to the reaction solution, and stir at room temperature for 3 hours after the addition is complete. Concentrate the reaction solution under reduced pressure, and purify the crude compound 12 by high performance liquid chromatography (separation conditions: column: Sharpsil-T C18 5 μm 21.2*250 mm; mobile phase: A-water (10 mmol _NH4OAc ), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to obtain the title product (7 mg, 15 mg).

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

Single-configuration compounds (shorter retention time)

UPLC analysis: retention time 1.345 min, purity: 72% (column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

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

Single-configuration compounds (longer retention time)

UPLC analysis: retention time 1.399 min, purity: 88% (column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.36(d,1H),7.77(d,1H),7.31(s,1H),6.51(s,1H),5.58-5.51(m,1H),5. 48(d,1H),5.42(s,1H),5.20(q,2H),4.09-4.02(m,1H),3.22-3.11(m,2H),2.3 9(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 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]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

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

Example 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide14-B

first step

3-Cyclopropyl-2-hydroxypropanoate benzyl ester 14a

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

Step 2

10-(cyclopropylmethyl)-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 14b

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

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

Step 3

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

14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, and palladium on carbon (7 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to obtain the crude product 14c (23 mg). The product was directly used in the next step of the reaction without further purification.

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

Step 4

(9H-fluorene-9-yl)methyl(2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropyl-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

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

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

Step 5

2-((2-aminoacetamido)methoxy)-3-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)propionamide 14e

14d (29 mg, 33.9 μmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted. This process was repeated three times. The residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 14e (22 mg). The product was used directly in the next reaction without purification.

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

Step 6

N-((2R,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide14-B

Crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. Then, 8 g (24 mg, 50.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (14 mg, 50.6 μmol) were added sequentially. The ice bath was removed, and the mixture was brought to room temperature and stirred for 1 hour to produce compound 14. The reaction solution was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH₄OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min) to obtain the title product (2 mg, 2 mg).

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

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.18 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.23 min, purity: 96% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

Example 15

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 15

first step

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid benzyl ester 15b

Add 8b (500 mg, 1.35 mmol) to the reaction flask, add 6 mL of tetrahydrofuran, and add 15a of 1-hydroxymethylcyclopropane-1-carboxylic acid (233 mg, 1.13 mmol; prepared by the method disclosed in Example 22-2 on page 262 of patent application “EP2862856A1”). Purge three times with argon, cool to 0-5°C in an ice-water bath, add sodium hydride (54 mg, 1.35 mmol), remove the ice bath, and stir at room temperature for 40 minutes. Cool to 0°C and add 20 mL of ice water. Extract with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). Combine the organic phases, wash with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system B to obtain the title product 15b (15 mg, yield: 2.5%).

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

Step 2

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

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

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

Step 3

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

Add 1b (10 mg, 0.021 mmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add 15c (11 mg, 0.026 mmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (10.7 mg, 0.039 mmol), and stir the reaction at room temperature for 60 minutes after the addition is complete. Add 10 mL of water, extract with ethyl acetate (5 mL × 3), wash the organic phase with saturated sodium chloride solution (10 mL), dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by thin-layer chromatography with developing solvent system B to give the title product 15d (19 mg, yield 87.0%).

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

Step 4

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

15d (19 mg, 22.56 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure at 0 °C, and 1 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the upper n-hexane layer was decanted. This process was repeated three times. The crude product 15e (13.9 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

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

Step 5

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 15

Crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (15.8 mg, 33.4 μmol) was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (9.3 mg, 33.6 μmol) was added, and the mixture was stirred at room temperature for 60 minutes. The reaction solution was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product 15 (2.5 mg, yield: 10.3%).

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

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

Example 16

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-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]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 16

first step

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

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

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

Step 2

1-(hydroxymethyl)cyclobutane-1-carboxylic acid benzyl ester 16c

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

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

Step 3

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid benzyl ester 16d

Add 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) to the reaction flask, add 3 mL of tetrahydrofuran, purge three times with argon, cool to 0-5 °C in an ice-water bath, add potassium tert-butoxide (61 mg, 0.54 mmol), and stir in an ice-water bath for 10 minutes. Add 20 mL of ice water, extract with ethyl acetate (5 mL) and chloroform (5 mL × 5), combine the organic phases and concentrate. Dissolve the residue in 3 mL of 1,4-dioxane, add 0.5 mL of water, add sodium bicarbonate (27 mg, 0.32 mmol) and fluorenemethyl chloroformate (71 mg, 0.27 mmol), and stir at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system C to give the title product 16d (24 mg, yield: 16.7%).

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

Step 4

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

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

Step 5

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclobutyl)methoxy)methyl)amino)2-oxoethyl)carbamate 16f

Add 1b (7.5 mg, 0.014 mmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add 0.5 mL of crude 16e (10 mg) in N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (6 mg, 0.026 mmol), and stir in an ice bath for 30 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (10 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by thin-layer chromatography using solvent system B to give the title product 16f (10.6 mg, yield 87.8%).

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

Step 6

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

16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product was concentrated under reduced pressure to obtain 16 g (8 mg) of the title product. The product was used directly in the next reaction without purification.

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

Step 7

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-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]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 16

16 g (8 mg) of the crude product was dissolved in 1 mL of N,N-dimethylformamide, and 8 g (8.8 mg, 18.6 μmol) was added. Then, 5.2 mg (18.8 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH₄OAc ): 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 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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12,15-pentoxo-17,20,23,26,29,32,35,38,41-nonoxa-2,5,8,11,14-pentazatritoxy-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 17

first step

1-Phenylacetic-2,5,8,11,14,17,20,23,26,29-decaoxane-31-tert-butyl ester 17b

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

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

Step 2

29-Hydroxy-3,6,9,12,15,18,21,24,27-Nineoxa-2,9-nona-1-octa-tert-butyl ester 17c

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

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

Step 3

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

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

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

Step 4

29-Amino-3,6,9,12,15,18,21,24,27-nonoxa-2,9-dioxo-1-acid tert-butyl ester 17e

17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, and palladium on carbon (58 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to obtain the crude product 17e (220 mg). The product was directly used in the next step of the reaction without further purification.

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

Step 5

1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonoxa-2-aza-tetrazol-31-oic acid tert-butyl ester 17f

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

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

Step 6

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

17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 1 hour. 15 mL of dichloromethane was added for dilution, and the mixture was concentrated under reduced pressure. 10 mL of n-hexane was added, and the mixture was concentrated under reduced pressure, repeated twice. 10 mL of toluene was added, and the mixture was concentrated under reduced pressure. The mixture was then slurried three times with a 5:1 mixture of n-hexane and diethyl ether until the pH was close to 7. The solution was concentrated and dried using an oil pump to give 17 g (98 mg, yield: 86.8%) of the title product.

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

Step 7

2,4-Dimethoxybenzyl-1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropyl-1-carboxylic acid ester 17h

Dissolve 8d (164 mg, 0.40 mmol) in dichloromethane (5 mL), then add 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.60 mmol), and 4-dimethylaminopyridine (5 mg, 0.041 mmol) sequentially. After addition, stir at room temperature for 1 hour. Add 20 mL of water, shake, and separate the layers. Extract the aqueous phase with dichloromethane (8 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography with solvent system C to give the title product 17h (124 mg, yield: 55.4%).

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

Step 8

2,4-Dimethoxybenzyl(S)-1-((11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazahepta-17-yl)oxy)cyclopropyl-1-carboxylic acid ester 17j

Dissolve 17h (39 mg, 69.6 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 1 hour. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene, and concentrate under reduced pressure twice; add 3 mL of n-hexane, stir, and decant the upper n-hexane layer, repeat three times, and concentrate under reduced pressure. Dissolve the obtained crude product in 2 mL of N,N-dimethylformamide, add (((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared using the method disclosed in Examples 7-12 on page 13 of patent application CN108853514A), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (23 mg, 83.1 μmol), and stir at room temperature for 1 hour. Add 10 mL of water and extract with ethyl acetate (10 mL × 3). Combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue 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].

Step 9

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

17kJ (48 mg, 58.4 μmol) was dissolved in 1.4 mL of a 3% (v/v) dichloroacetic acid solution in dichloromethane. The solution was cooled to 0–5 °C in an ice-water bath, and triethylsilane (21 mg, 180.6 μmol) was added. The mixture was stirred in an ice bath for 3 hours. The solution 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 mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried using an oil pump to give the title product 17k (33 mg, yield: 84.1%).

Step 10

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

Add 1b (20 mg, 42.4 μmol) to the reaction flask, then add 1 mL of 10% (v/v) methanol in dichloromethane solution. Purge three times with argon gas, cool in an ice-water bath to 0–5 °C, and add one drop of triethylamine, stirring until 1b dissolves. Dissolve 17k (33 mg, 49.1 μmol) in 1 mL of 10% (v/v) methanol in dichloromethane solution, then add it dropwise to the above reaction solution, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (17.6 mg, 63.6 μmol). Heat to room temperature and stir for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, allow to stand for separation, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 17l (37 mg, yield: 80.2%).

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

Step 11

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

17 L (15.5 mg, 14.23 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the upper n-hexane layer was decanted. This process was repeated three times. The mixture was concentrated under reduced pressure and then dried using an oil pump. The crude product was dissolved in 1 mL of N,N-dimethylformamide, and 17 g (11 mg, 15.92 μmol) was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (6.0 mg, 21.68 μmol) was added. The mixture was purged with argon three times, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 17 (6mg, yield: 27.4%).

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

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.98(d,1H),8.76(s,1H),8.20(br,1H),8.12-7.95(m,3H),7.93-7.76(m,2H),7.75-7.66(m,2H),7.24(s,1H),7.20-7.05(m,6H),6.9 7(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,1 H),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),3.1 5-3.08(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 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]indolazino[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-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 18

first step

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

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

2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile, and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 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 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue (4.1 g) was purified by silica gel column chromatography with solvent C as the developing solvent. Further chiral resolution and purification yielded the title products 18a (1.1 g) and 18b (1.2 g).

Step 2

(R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaun-11-acid benzyl ester

18c

Dissolve 8b (3.1 g, 8.41 mmol) in tetrahydrofuran (55 mL), add 18a (2.0 g, 9.70 mmol), cool to 0–5 °C in an ice-water bath, add potassium tert-butoxide (1.89 g, 16.84 mmol), and stir in an ice-water bath for 10 minutes. Add ethyl acetate (30 mL) and water (20 mL), allow to stand for separation, extract the aqueous phase with chloroform (30 mL × 5), and combine the organic phases. Concentrate the organic phase under reduced pressure, dissolve the residue in 1,4-dioxane (32 mL) and water (8 mL), add sodium carbonate (1.78 g, 16.79 mmol) and fluorenyl chloroformate (2.18 g, 8.42 mmol), and stir at room temperature for 2 hours. Add water (30 mL) to the reaction solution, extract with ethyl acetate (50 mL × 3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with developing solvent system C to give the title product 18c (1.3 g, yield: 30.0%).

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

Step 3

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

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

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

Step 4

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

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

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

Step 5

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

18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product 18f (21 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

Step 6

2,4-Dimethoxybenzyl(11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-ester 18g

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

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

Step 7

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

18 g (37 mg, 44.3 μmol) was dissolved in 1.4 mL of 3% (v/v) dichloroacetic acid in dichloromethane solution. The solution was cooled to 0-5 °C in an ice-water bath, and triethylsilane (15.4 mg, 132.4 μmol) was added. The mixture was stirred in an ice bath for 3 hours. The solution 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 mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried under an oil pump to give the title product 18h (24 mg, yield: 79.1%). MS m/z (ESI): 708.2 [M+23].

Step 8

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexa-16-yl)carbamate 18i

Add 1b (30 mg, 63.6 μmol) to the reaction flask, add 1 mL of 10% (v/v) methanol in dichloromethane solution, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until 1b dissolves. Dissolve 18h (65 mg, 94.8 μmol) in 1 mL of 10% (v/v) methanol in dichloromethane solution, then add it dropwise to the above reaction solution, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (27 mg, 97.6 μmol). Heat to room temperature and stir for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, allow to stand for separation, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 18i (25 mg, yield: 35.6%).

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

Step 9

(S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropionamide18j

18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred. The upper n-hexane layer was decanted. This process was repeated three times. The crude product 18j (10 mg) was concentrated under reduced pressure and used directly in the next reaction step without purification.

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

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

Crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17 g (8.5 mg, 12.3 μmol) was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.6 mg, 16.6 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was filtered and purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD5um 19*250mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain title product 18 (9.5 mg, yield: 56.2%).

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

¹ H NMR (400 MHz, DMSO-d ₆ ): δ8.77(d,1H),8.59-8.55(m,1H),8.42(d,1H),8.37-8.28(m,1H),8.25-8.06(m,2H),7.96-7.86(m,1H),7.86-7.70(m,2H) ,7.32-7.28(m,1H),7.25-7.14(m,3H),6.67(m,1H),5.96(s,1H),5.80-5.72(m,1H),5.62-5.52(m,2H),5.43-5.30(m,3H),5. 28-5.17(m,2H),5.12-5.08(m,1H),4.72-4.35(m,8H),3.95-3.70(m,13H),3.35-3.22(m,14H),2.42-2.32(m,3H),2.05-1.98 (m,4H),1.88-1.82(m,12H),1.47-1.39(m,3H),1.32-1.18(m,11H),0.90-0.80(m,4H),0.52-0.37(m,3H),0.32-0.18(m,2H).

Example 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]indolazino[1,2-b]quinoline (Lin-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-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 19

first step

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

Add 18b (252 mg, 1.22 mmol) to the reaction flask, add 4 mL of dichloromethane, purge three times with argon, cool to 0-5 °C in an ice-water bath, add lithium tert-butoxide (98 mg, 1.22 mmol), stir in an ice-water bath for 15 minutes until clear, add 8b (300 mg, 814.3 μmol), stir in an ice-water bath for 2.5 hours. Add water (10 mL), separate the layers, extract the aqueous phase with dichloromethane (8 mL × 2), combine the organic phases, wash with water (10 mL × 1), wash with saturated brine (10 mL × 2), dry with anhydrous sodium sulfate, filter and concentrate to obtain crude product. Purify the residue by silica gel column chromatography with solvent system C to obtain title product 19a (282 mg, yield: 67.2%).

Step 2

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

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

Step 3

2,4-Dimethoxybenzyl(S)-10-cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabutane-11-ester 19c

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

Step 4

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

19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, and 0.5 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product 19d (21 mg) was concentrated under reduced pressure and used directly in the next reaction step without purification.

Step 5

2,4-Dimethoxybenzyl(11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo

-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-ester 19e

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

Step 6

(11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2,18-dioxane

-4,7,10,13,16-pentazazatetra-20-acid 19f

19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of a 3% (v/v) dichloroacetic acid solution in dichloromethane. The solution was cooled to 0–5 °C in an ice-water bath, and triethylsilane (29 mg, 249.4 μmol) was added. The mixture was stirred in an ice bath for 3 hours. The solution 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 mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried using an oil pump to give the title product 19f (57 mg, yield: 99.2%).

Step 7

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexa-16-yl)carbamate 19g

Add 1b (30 mg, 63.6 μmol) to the reaction flask, then add 1 mL of 10% (v/v) methanol in dichloromethane solution. Purge three times with argon gas, cool to 0-5 °C in an ice-water bath, and add one drop of triethylamine, stirring until 1b dissolves. Dissolve 19f (57 mg, 83.1 μmol) in 1 mL of 10% (v/v) methanol in dichloromethane solution, then add it dropwise to the above reaction solution, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (26 mg, 93.9 μmol). Heat to room temperature and stir for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, allow to stand for separation, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL × 3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give 19 g (56 mg, yield: 79.8%) of the title product.

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

Step 8 (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide 19h

19 g (4.6 mg, 4.16 μmol) was dissolved in 1.5 mL of dichloromethane, and 0.75 mL of diethylamine was added. The mixture was stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product 19 h (4.0 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

Step 9

(1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline (Lin-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-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 19

The crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17 g (2.9 mg, 4.2 μmol) was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (1.5 mg, 5.4 μmol) was added, and the mixture was stirred at room temperature for 40 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5um 19*250 mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, yield: 32.4%).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ8.71-8.62(m,1H),8.59-8.51(m,1H),8.34-8.26(m,1H),8.14-8.02 (m,2H),7.95-7.86(m,1H),7.83-7.69(m,2H),7.35-7.31(m,1H),7.29-7 .11(m,3H),7.01(s,1H),6.72-6.50(m,3H),5.59-5.50(m,2H),5.42(s,2 H),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 20 (Reference Example)

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

The following antibodies were prepared using standard antibody methods, such as constructing a vector, transfecting eukaryotic cells like HEK293 cells (Life Technologies Cat. No. 11625019), and purifying and expressing them.

The following is the sequence of Trastuzumab:

Light chain

DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.1

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.2

The following is the sequence of Pertuzumab:

Light chain

DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.3

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.4

The following is the sequence of B7H3 antibody 1F9DS:

Light chain

DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKL TVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

SEQ ID NO.5

Heavy chain

QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQG ALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 21 ADC-1

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

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

UV-HPLC calculated average: n = 5.09.

Example 22 ADC-2

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

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

UV-HPLC calculated average: n = 7.39.

Example 23 ADC-3

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

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

UV-HPLC calculated average: n = 7.36.

Example 24 ADC-4

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.74 mL, 13.38 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 6.7 mg/mL, and 1.3 mL of the solution was taken out and used for further reaction.

Compound 9-A (1.0 mg, 0.93 μmol), a compound with a shorter retention time, was dissolved in 0.10 mL of DMSO and added to the above 1.3 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.72 mg/mL, 2.36 mL) of the exemplary product ADC-4 of the general formula FADC-4A, which was stored frozen at 4 °C.

UV-HPLC calculated average: n = 7.39.

Example 25 ADC-5

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) was added to the PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.0 mL, 6.70 mg/mL, 0.136 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, and 0.614 mL of the solution was taken out and the reaction was continued.

Compound 9-A (0.5 mg, 0.42 μmol), a compound with a shorter retention time, was dissolved in 0.031 mL of DMSO and added to the above 0.614 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (3.08 mg/mL, 0.82 mL) of the exemplary product ADC-5 of the general formula FADC-4A, which was stored frozen at 4 °C.

UV-HPLC calculated average: n = 3.16.

Example 26 ADC-6

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.74 mL, 13.38 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 6.7 mg/mL, and 0.75 mL of the solution was taken out and used for further reaction.

Compound 9-B (0.68 mg, 0.63 μmol), a compound with a longer retention time, was dissolved in 0.10 mL of DMSO and added to the above 0.75 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.78 mg/mL, 1.78 mL) of the exemplary product ADC-6 of the general formula FADC-4B, which was stored frozen at 4 °C.

UV-HPLC calculated average: n = 3.94.

Example 27 ADC-7

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 1.0 mL of the solution was taken out and used for further reaction.

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

UV-HPLC calculated average: n = 6.91.

Example 28 ADC-8

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 1.6 mL of the solution was taken out and used for further reaction.

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

UV-HPLC calculated average: n = 6.58.

Example 29 ADC-9

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 0.8 mL of the solution was taken out and used for further reaction.

Compound 9-A (0.55 mg, 0.5 μmol), a compound with a shorter retention time, was dissolved in 0.1 mL of DMSO and added to the above 0.8 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.27 mg/mL, 1.11 mL) of the exemplary product ADC-9 of the general formula FADC-9A, which was stored frozen at 4 °C.

UV-HPLC calculated average: n = 3.16.

Example 30 ADC-10

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/ml, 0.574 mL, 38.78 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then 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 added to the above reaction solution. The mixture was placed in a water bath and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (5.48 mg/mL, 1.03 mL) of the exemplary product ADC-10 of the general formula FADC-10, which was stored frozen at 4°C.

UV-Vis calculated average: n = 6.25.

Example 31 ADC-11

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 0.646 mL, 43.64 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.03.

Example 32 ADC-12

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 uL, 250.0 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 0.726 mL, 49.05 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.93.

Example 33 ADC-13

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/ml, 0.287 mL, 19.39 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.53.

Example 34 ADC-14

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 0.592 mL, 40.0 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.61.

Example 35 ADC-15

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 0.592 mL, 40.0 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.89.

Example 36 ADC-16

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 0.53 mL, 35.8 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.43.

Example 37 ADC-17

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 uL, 432 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 2.0 mL, 135.12 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 5.42.

Example 38 ADC-18 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 uL, 517 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.5 mL, 101.3 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.23.

Example 39 ADC-19

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.36 mL, 91.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.26.

Example 40ADC-20

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 uL, 517 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.5 mL, 101.3 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.43.

Example 41 ADC-21 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.86 mL, 125.4 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.23.

Example 42 ADC-22

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 uL, 649 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.88 mL, 127.2 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.79.

Example 43 ADC-23 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 345 mL, 23.31 μol). The solution was placed in a water bath and shaken at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified by ultrafiltration membrane successively with PBS buffer solution (0.05 M PBS buffer solution, pH = 6.5) containing 2% (v/v) MeCN and 1% (v/v) DMSO, and succinate buffer solution (0.01 M succinate buffer solution, pH = 5.3). Sucrose was then added to a concentration of 60 mg/mL and Tween 20 to a concentration of 0.2 mg/mL. The mixture was bottled and lyophilized to obtain the lyophilized powder sample of the exemplary product ADC-23 of the general formula FADC-18, which was stored frozen at 4 °C.

UV-Vis calculated average: n = 7.05.

Example 44 ADC-24

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 332 mL, 22.43 μol). The solution was placed in a water bath and shaken at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (241 mg, 224 μmol), a compound with a shorter retention time, was dissolved in 13.76 mL of MeCN and 6.88 mL of DMSO. This solution was added to the above reaction mixture, and the mixture was placed in a water bath and shaken at 25 °C for 3 hours. The reaction was then stopped. The reaction mixture was purified by ultrafiltration using a PBS buffer solution containing 4% (v/v) MeCN and 2% (v/v) DMSO (0.05 M PBS buffer solution, pH = 6.5) and a succinate buffer solution (0.01 M succinate buffer solution, pH = 5.3). Sucrose was then added to a concentration of 60 mg/mL, and Tween 20 was added to a concentration of 0.2 mg/mL. The mixture was then lyophilized in bottles to obtain the lyophilized powder sample of the exemplary product ADC-24 of the general formula FADC-4A, which was stored frozen at 4 °C.

UV-Vis calculated average: n = 7.07.

Example 45 ADC-25

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 uL, 740 nmol) was added to a PBS buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/ml, 2.14 mL, 144.60 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.87.

Example 46 ADC-26 (Reference Example)

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

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

UV-Vis calculated average: n = 6.15.

Example 47 ADC-27

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

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

UV-Vis calculated average: n = 6.11.

Example 48 ADC-28 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 uL, 810 nmol) was added to a PBS-buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/ml, 2.36 mL, 159.47 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.46.

Example 49 ADC-29

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffer solution; 10.0 mg/mL, 0.80 mL, 50.06 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 7.24.

Example 50 ADC-30 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) was added to a PBS-buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/ml, 0.86 mL, 58.4 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

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

UV-Vis calculated average: n = 6.15.

Example 51 ADC-31

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

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

UV-Vis calculated average: n = 6.33.

ADC drug loading analysis

Experimental Objectives and Principles

Antibody-associated drug (ADC) stock solution is a type of antibody-crosslinked drug. Its therapeutic mechanism relies on the targeted delivery of toxin molecules into cells, thereby killing the cells. Drug loading plays a decisive role in efficacy. The drug loading of the ADC stock solution was determined using ultraviolet light.

Experimental methods

After placing cuvettes containing sodium succinate buffer solution into the reference absorption cell and the sample absorption cell, respectively, and subtracting the solvent blank, cuvettes containing the test solution were placed into the sample absorption cell, and absorbance was measured at 280 nm and 370 nm.

Results Calculation: The ADC stock solution loading was determined using ultraviolet spectrophotometry (instrument: Thermo Nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength, i.e.:

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

ε _Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm;

C _Drug : Drug concentration;

ε _mab-280 : Trastuzumab antigen solution or pertuzumab antigen solution has an average molar extinction coefficient of 214600 at 280 nm;

C _mab : Concentration of trastuzumab antigen solution or pertuzumab antigen solution;

b: The optical path length is 1cm.

Similarly, the equation for the total absorbance of the sample at 370 nm can be obtained:

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

ε _Drug-370 : The drug has an average molar extinction coefficient of 19000 at 370 nm;

C _Drug : Drug concentration;

ε _mab-370 : Trastuzumab antigen solution or pertuzumab antigen solution has an extinction coefficient of 0 at 370nm;

C _mab : Concentration of trastuzumab antigen solution;

b: The optical path length is 1cm.

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

Drug load = C_{_Drug} / C_{_mab} .

Biological evaluation

Test Example 1: In vitro proliferation inhibition test of compounds of general formula (D) on tumor cells

I. Purpose of the Test

The purpose of this experiment was to detect the inhibitory activity of the drug compound of general formula (D) of this invention on the in vitro proliferation of U87MG cells (Chinese Academy of Sciences Cell Bank, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). Cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, cell proliferation was detected using the CTG (Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent. The in vitro activity of the compound was evaluated based on the IC50 value.

II. Experimental Methods

The following example, using the in vitro proliferation inhibition assay of U87MG cells, illustrates the method for testing the in vitro proliferation inhibition activity of the compounds of this invention against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

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

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

3. Cell Plating: Mix U87MG and SK-BR-3 single-cell suspensions thoroughly. Adjust the viable cell density to 2.75 × ^10³ cells/ml and 8.25 × ^10³ cells/ml respectively using cell culture medium. Mix the adjusted cell suspensions thoroughly and add 180 μl/well to a 96-well cell culture plate. Add only 200 μl of culture medium to the outer wells of the 96-well plate. Incubate the plates in an incubator for 24 hours (37°C, 5% _CO₂ ).

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

The initial concentration of the small molecule compound is 500 nM, and the preparation method is as follows.

Add 30 μl of different test samples (100 μM) to each well in the first column of a 96-well U-bottom preparation plate. Add 20 μl of DMSO to each well in columns 2 through 11. Transfer 10 μl of the sample from the first column to the 20 μl of DMSO in the second column, mix well, and then transfer 10 μl to the third column, and so on up to the tenth column. Transfer 5 μl of the drug from each well of the preparation plate to 95 μl of EMEM medium, mix well, and set aside for later use.

The initial concentration of the ADC is 10 nM or 500 nM, and the preparation method is as follows.

Add 100 μl of different test samples to the first column of a 96-well plate, with a sample concentration of 100 nM or 5 μM. Add 100 μl of PBS to each well in columns 2 through 11. Take 50 μl of the sample from the first column and add it to the 100 μl of PBS in the second column, mix well, take 50 μl and add it to the third column, and so on, diluting 3-fold up to the 10th column.

5. Sample addition procedure: Add 20 μl of the prepared test sample at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% _CO2 ).

6. Color development procedure: Take out the 96-well cell culture plate, add 90 μl of CTG solution to each well, and incubate at room temperature for 10 minutes.

7. Plate reading procedure: Take out the 96-well cell culture plate and place it in an ELISA reader (BMG Labtech, PHERAstar FS) to measure chemiluminescence.

III. Data Analysis

The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in the table below.

Table 1 shows the _IC50 values of the small molecule fragments disclosed in this publication on the in vitro proliferation inhibition of SK-BR-3 and U87 cells.

Conclusion: The small molecule fragments disclosed in this study exhibit significant inhibitory activity against the proliferation of SK-BR-3 and U87 cells, and the chiral center has a certain influence on the inhibitory activity of the compound.

Test Example 2: In vitro proliferation inhibition test of the antibody-drug conjugate of this disclosure on HER2-targeted tumor cells

The purpose of this experiment was to detect the inhibitory activity of the antibody-drug conjugate targeting HER2 disclosed herein on the in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, catalog number HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, catalog number HTB-132). Cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, cell proliferation was detected using CTG reagent. The in vitro activity of the compound was evaluated based on the _IC50 value.

Following the test method in Test Example 1, the test cells were SK-BR-3 and MDA-MB-468. The cell culture media were McCoy's 5A medium (Gibco, catalog number 16600-108) containing 10% FBS, EMEM medium (GE, catalog number SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, catalog number 11415-114) containing 10% FBS, respectively. The viable cell densities of the three cell lines were adjusted to 8.33 × ^10³ cells/ml, ^{8.33 × 10³} cells/ml, and 1.39 × ^10⁴ cells/ml, respectively. The density-adjusted cell suspensions were thoroughly mixed, and 180 μl/well was added to a 96-well cell culture plate. The relevant compounds were tested, and the results are shown in the table below.

Table 2 discloses the _IC50 values of antibody-drug conjugates on the in vitro proliferation inhibition of HER2-targeting tumor cells.

Conclusion: The antibody-drug conjugates targeting HER2 disclosed in this study have significant inhibitory activity against the proliferation of HER2-positive SK-BR-3 cells; at the same time, they have weak inhibitory activity against the proliferation of HER2-negative MDA-MB-468 cells, demonstrating good selectivity.

Test Example 3: Her2-ADC Plasma Stability Experiment

ADC-19, ADC-18, ADC-20 samples, human plasma, monkey plasma (Shanghai Medicilon Biopharmaceutical Co., Ltd.), and 1% BSA (Sigma) PBS solution (Shanghai Sangon Biotech) were sterilized by filtration through a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the sterile plasma or 1% BSA PBS solution at a final concentration of 200 μg/ml and incubated at 37°C in a cell culture incubator. The day of incubation was designated as day 0. Samples were then collected on days 7, 14, and 21 for free toxin detection.

Transfer 25 μl of sample to a 96-well plate; add 50 μL of internal standard working solution (100 ng/mL camptothecin acetonitrile solution) and 150 μl of acetonitrile; vortex for 5 minutes, centrifuge for 10 minutes (4000 rpm), and perform LC/MS/MS (Applied Biosystems, Inc.) analysis in 5 μL.

The results showed that ADC-19 was quite stable in human and monkey plasma as well as in 1% BSAPBS solution, with the release rate of free toxin not exceeding 2.1% and stabilizing by day 14 (Figure 1A).

ADC-18 exhibits poor stability in human and monkey plasma, with the highest release rates of free toxins reaching 14.5% and 8.10%, respectively. It is relatively stable in 1% BSA PBS solution (see Figure 1B).

ADC-20 exhibited poor stability in human plasma, monkey plasma, and 1% BSA PBS solution, with the highest release rates of free toxins at 21.7%, 29.7%, and 21.7%, respectively. Furthermore, it remained in a degrading state in 1% BSA PBS solution (see Figure 1C).

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

I. Experimental Objective

Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 administered intraperitoneally in nunu mice with trastuzumab-resistant human breast cancer cell line JIMT-1 xenografts.

II. Test Drugs and Materials

1. Test drug

T-DM1 (prepared according to US20050169933)

ADC-21: 3 mg/kg

ADC-21: 10 mg/kg

ADC-24: 3 mg/kg

ADC-24: 10 mg/kg

Blank control: PBS

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

nunu nude mouse, purchased from Beijing Vital River.

III. Test Methods

JIMT-1 cells (Nanjing Kebai) (5× ^10⁶ /mouse, with 50% artificial basement membrane) were subcutaneously injected into the right rib area of mice. After 8 days of tumor growth, when the tumor reached 203.09±11.94 ^mm³ , the animals were randomly divided into 6 groups (d1), with 8 mice per group.

The medication was administered via intraperitoneal injection, twice in total. Tumor volume and body weight were measured twice weekly, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 2. The drug was administered intraperitoneally twice, and the experiment ended on day 34. T-DM1 (10 mg/kg) had no inhibitory effect on tumors; ADC-21, 3 mg/kg, showed a tumor inhibition rate of 46.22% (P < 0.01); ADC-21, 10 mg/kg, showed a tumor inhibition rate of 56.77% (P < 0.001); ADC-24, 3 mg/kg, showed a tumor inhibition rate of 62.77% (P < 0.001); ADC-24, 10 mg/kg, showed a tumor inhibition rate of 76.32% (P < 0.001). At the same dosage, ADC-24 showed significantly better tumor inhibition than ADC-21.

Test Example 5: Evaluation of the efficacy of SK-BR-3 tumor-bearing mice

I. Experimental Objective

Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies ADC-21 and ADC-22 administered intraperitoneally on nunu mice with human breast cancer cell xenograft SK-BR-3.

II. Test Drugs and Materials

1. Test drug

ADC-21: 1 mg/kg

ADC-21: 6 mg/kg

ADC-22: 1 mg/kg

ADC-22: 6 mg/kg

Blank control: PBS.

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

nunu nude mouse, purchased from Beijing Vital River.

III. Test Methods

SK-BR-3 cells (ATCC) (5× ^10⁶ /mouse, with 50% artificial basement membrane) were subcutaneously injected into the right rib area of mice. After 20 days of tumor growth, when the tumor reached 153.34±11.73 mm³, the animals were randomly divided into 5 groups (d0), with 8 mice per group.

The medication was administered once via intraperitoneal injection. Tumor volume and body weight were measured twice weekly, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control and experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 3. After a single intraperitoneal injection, the experiment ended on day 28. The tumor inhibition rate of ADC-21 at 1 mg/kg was 15.01%; the tumor inhibition rate of ADC-21 at 6 mg/kg was 77.4%, showing a highly significant difference compared to the blank control (P < 0.001). The tumor inhibition rate of ADC-22 at 1 mg/kg was 19.82%; the tumor inhibition rate of ADC-22 at 6 mg/kg was 98.38% (P < 0.001). At the same dose of 6 mg/kg, the tumor inhibition effect of ADC-22 was significantly better than that of ADC-21.

Test Example 6: Plasma Stability

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

After samples were taken out at different time points, they were placed at room temperature and vortexed to mix. 25 μl of the sample was transferred to a 96-well plate. 50 μL of internal standard working solution (100 ng/mL camptothecin acetonitrile solution) and 150 μl of acetonitrile were added. The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 5 μl of the supernatant was taken for LC/MS/MS analysis.

As shown in Figure 4, ADC-25 was quite stable in human and monkey plasma as well as in 1% BSAPBS solution, with the release rate of free toxin not exceeding 2% and stabilizing by day 14.

Test Example 7: Evaluation of the efficacy of ADC in human astrocytoma U87MG xenografts in nude mice I. Experimental Objective

This experiment used BALB/cA-nude nude mice as test animals to evaluate the efficacy of the disclosed ADC compound on human astrocytoma U87MG nude mouse xenografts.

II. Test Drugs and Materials

1. Test drug

ADC-27 (3 mg/kg)

ADC-26 (3 mg/kg)

Blank control: PBS buffer at pH 7.4.

2. Preparation method: PBS buffer with pH 7.4.

3. Experimental animals

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

III. Test Methods

Female BALB/cA-nude nude mice, 6-7 weeks old, were subcutaneously inoculated with human astrocytoma U87MG cells (human astrocytoma, Chinese Academy of Sciences Cell Bank, Catalog#TCHu138). Ten days after cell inoculation, the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections of the drug were administered once weekly for a total of 3 times. Tumor volume and body weight were measured 2-3 times per week, and the data were recorded. The formula for calculating tumor volume (V) is:

V = 1/2 × a × b ²

Where a and b represent length and width, respectively.

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.

IV. Test Results

Intraperitoneal injection (i.p.) was administered once weekly for a total of three times. By day 22, the tumor inhibition rate of ADC-27 3 mg/kg reached 63.3% (P<0.0001), while the inhibition rate of ADC-26 3 mg/kg reached 49.1%. ADC-27 showed stronger antitumor efficacy than ADC-26.

During the administration process, the weight of animals in all groups remained normal, indicating that the ADC had no obvious toxic side effects. The test results are shown in Table 3 and Figure 5. The tested antibody effectively inhibited the growth of U87MG xenografts in tumor-bearing nude mice in a dose-dependent manner.

Table 3. Efficacy of the administered antibody against human astroblastoma U87MG xenografts in nude mice (D22)

Test Example 8: Evaluation of the efficacy of ADC in Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases

I. Experimental Objective

This experiment used BALB/cA-nude nude mice as test animals to evaluate the efficacy of the disclosed ADC compound against Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

II. Test Drugs and Materials

1. Test drug

ADC-29 (3 mg/kg)

ADC-28 (3mg/kg)

Negative control ADC (3 mg/kg): a ligand toxin conjugate formed by coupling non-B7H3 target with compound 20.

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

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

III. Test Methods

Female BALB/cA-nude nude mice, 6-7 weeks old, were subcutaneously inoculated with Detroit 562 cells (ATCC, Catalog#CCL-138 ^™ ) that metastasize to human pharyngeal carcinoma in pleural effusion. On day 10 post-inoculation, the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injection of the drug was initiated once weekly for a total of 3 times. Tumor volume and body weight were measured 2-3 times weekly, and data were recorded. The formula for calculating tumor volume (V) is:

V = 1/2 × a × b ²

Where a and b represent length and width, respectively.

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The drugs were administered intraperitoneally once a week for a total of three times. By day 28, the tumor inhibition rates of the tested ADCs were as follows: ADC-29 3 mg/kg (3 MPk) achieved a tumor inhibition rate of 72.27% (P<0.001); ADC-28 3 mg/kg (3 MPk) achieved a tumor inhibition rate of 56.2% (P<0.001). ADC-29 showed stronger antitumor efficacy than ADC-28 in both cases.

During the administration process, the weight of animals in all groups remained normal, indicating that the ADC had no obvious toxic side effects. The test results are shown in Table 4 and Figure 6. The tested antibody effectively inhibited the growth of Detroit 562 xenografts in tumor-bearing nude mice in a dose-dependent manner.

Table 4. Efficacy of antibody administration on Detroit 562 xenografts in tumor-bearing nude mice (D28)

Test Example 9: Efficacy Evaluation of U87-MG Tumor-Bearing Mice

I. Experimental Objective

Using Balb/c nude mice as test animals, the efficacy of intraperitoneal injection of B7H3-antibody-drug conjugates was evaluated in a human glioma cell U87MG xenograft model.

II. Test Drugs and Materials

1. Test drug

ADC-30 1mg/kg

ADC-30 3mg/kg

ADC-31 1mg/kg

ADC-31 3mg/kg

Blank control: PBS

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

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

III. Test Methods

U87MG cells (human astroblastoma, Chinese Academy of Sciences Cell Bank, Catalog#TCHu138) (2.5× ^10⁶ /mouse) were subcutaneously injected into the right rib area of mice. After the tumor grew for 14 days and reached 167.49 mm³, the animals were randomly divided into 5 groups of 8 mice per group (d1).

The medication was administered via intraperitoneal injection once a week for a total of three times. Tumor volume and body weight were measured twice a week, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 7. After intraperitoneal injection once a week for a total of three administrations, the tumor inhibition rates of the tested ADCs were as follows by day 18: ADC-30 1 mg/kg: 0.31%; ADC-30 3 mg/kg: 45.23% (P<0.0001); ADC-31 1 mg/kg: 39.22% (P<0.01); ADC-31 3 mg/kg: 80.24% (P<0.0001). At the same dosage, ADC-31 showed significantly better tumor inhibition than ADC-30.

Sequence List

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Shanghai Hengrui Medicine Co., Ltd.

<120> Ligand-drug conjugates of eczema analogues, their preparation methods and applications

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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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

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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

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

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

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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

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

435 440 445

Gly Lys

450

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

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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

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

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

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

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

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<211> 449

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<221> Chain

<223> Pertuzumab heavy chain

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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 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

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<221> Chain

<223> B7H3 antibody 1F9DS light chain

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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

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> B7H3 antibody 1F9DS heavy chain

<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 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