Heterocyclic compound as well as preparation method and application thereof

Abstract

The invention relates to the field of medicine, in particular to a medicine linker compound, the medicine can comprise a DNA topoisomerase inhibitor and a conjugate prepared through the medicine linker compound, for example, the antibody-medicine conjugate has a better medicine-antibody coupling ratio and can be used for preparing a medicine-antibody conjugate. The compound has a good targeted killing effect on tumors such as lung cancer, melanoma, breast cancer, gastric cancer and colon cancer. In addition, the invention further provides a preparation method and application of the antibody drug-linker molecule.

Description

Heterocyclic compound and its preparation method and use Technical Field

[0001] The present application relates to the field of compounds, and in particular to a heterocyclic compound, which can be used to prepare conjugates, such as antibody-drug conjugates. Background Art

[0002] In the field of anti-cancer drug development, ADCs have attracted global attention for their targeted properties. ADCs are novel anti-cancer drugs that link a toxin drug to an antibody. Typically composed of an antibody, a toxin drug, and a linker, ADCs leverage the antibody's targeted recognition and the toxin's high activity to exert their anti-tumor effects, offering greater specificity and efficacy than traditional small molecule drugs.

[0003] After ADC enters the body, the toxin is transported to the tumor tissue through antibodies with antigen targeting. ADC binds to antigens on the surface of tumor cells, and the cells engulf the ADC, which is then broken down in lysosomes to release cytotoxins, destroying DNA or preventing cell division, thereby achieving the effect of killing cells.

[0004] The linker ensures the stability of the ADC in the blood, while the toxin exerts its killing effect after reaching the target. ADC toxins primarily include microtubule inhibitors, DNA-damaging agents, and RNA polymerase inhibitors. Linkers also include various structures, including cleavable and non-cleavable types. The structure of the toxin drug and linker is crucial to the efficacy and safety of the ADC. Therefore, the development of drug-linker compounds with excellent activity and safety remains a challenge in this field.

[0005] Summary of the Invention

[0006] The present application relates to a drug-linker compound and a preparation method and use thereof. The drug-linker compound can be used to form a conjugate with other molecules such as antibodies.

[0007] In one aspect, the present invention provides a drug-linker compound having the formula GM-[LED] x The structure shown, wherein:

[0008] G is a functional group or leaving group capable of reacting with specific amino acids or sugar groups;

[0009] M is a linker connected to G, and the M is

[0010] Wherein, ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkyne or amino, the C 1-20 Alkylene, C 2- 20 Alkenylene, C 2-20 The alkynylene or amine group is optionally substituted with one or more suitable substituents;

[0011] L is a linker between the linkers M and E, and L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs, and short peptides composed of amino acids,

[0012] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20;

[0013] E is a structural fragment connecting L and D, wherein E is a single bond, -NHCH2- or selected from the following structures:

[0014] D is a cytotoxic drug fragment; and / or

[0015] x is selected from 1 to 10.

[0016] In some embodiments, the G is a functional group or leaving group that can react with a specific amino acid or sugar group in an antibody.

[0017] In some embodiments, the G is selected from halogen, halogenated C 1-6 Alkyl, C 1-6 Sulfonyl, halo C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 Sulfoxide, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Sulfonyl, halo C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 The sulfoxide, alkenyl, alkynyl and alkynyl-containing structural fragments are optionally substituted with one or more suitable substituents.

[0018] In some embodiments, the G is selected from halogen, halogenated C 1-6 Alkyl, C 1-6 Sulfonyl, halo C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 Sulfoxide, nitro, azide, cyano, alkenyl, alkynyl and structural fragments containing alkynyl.

[0019] In some embodiments, the M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkyne or amino, the C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

[0020] In some embodiments, the M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene or amino.

[0021] In some embodiments, M is wherein ring A is selected from M1 is selected from a single bond and C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkyne or amino, the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

[0022] In some embodiments, the M is selected from

[0023] In some embodiments, the M is selected from

[0024] In some embodiments, the M is selected from

[0025] In some embodiments, the M is selected from

[0026] In some embodiments, L is selected from one or more of the following structures: 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) r OCH3)), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, V al-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gl y-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys -Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly,

[0027] Where R' represents hydrogen, C 1-6 an alkyl group or a polyethylene glycol fragment containing 1 to 10 EO units; s is selected from an integer of 1 to 20, for example, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0028] In some embodiments, L is selected from one or more of the following structures: 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1-20.

[0029] In some embodiments, L is selected from one or more of the following structures:

[0030] In some embodiments, L is selected from the following structures:

[0031] In some embodiments, L is selected from the following structures:

[0032] In some embodiments, L is selected from the following structures:

[0033] In some embodiments, L is selected from the following structures:

[0034] In some embodiments, E is a single bond, -NHCH2-, In some embodiments, E is a single bond.

[0035] In some embodiments, E is -NHCH2-.

[0036] In some embodiments, E is

[0037] In some embodiments, E is

[0038] In some embodiments, Selected from the following structures:

[0039] In some embodiments, Selected from the following structures:

[0040] In some embodiments, the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0041] In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound; the DNA intercalator is a pyrrolobenzodiazepine PBD; the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0042] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH2), secondary amine (-NR A H) or tertiary amine (-NR B R C ), where R A 、R B 、R C These represent only non-hydrogen substituents on N, through which the cytotoxic drug can be attached to the linker in the conjugate.

[0043] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through a -OH, -SH, primary amino group, secondary amine group, or tertiary amine group on the cytotoxic drug.

[0044] In some embodiments, the cytotoxic drug is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of a compound of Formula I or Formula II:

[0045] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0046] R3 is selected from H and -CO-CH2OH;

[0047] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0048] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0049] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0050] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0051] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0052] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0053] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0054] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0055] In some embodiments, the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug.

[0056] In some embodiments, D is selected from the following structures:

[0057] On the other hand, the present invention provides a drug linker compound having the formula GM-[LED] x The structure shown, wherein:

[0058] G is a functional group or leaving group capable of reacting with a specific amino acid or sugar group in an antibody or antigen-binding fragment; G is preferably selected from halogen, halogenated C 1-6 Alkyl, C 1-6 Sulfonyl, halo C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 Sulfoxide, nitro, azide, cyano, alkenyl, alkynyl, and structural fragments containing alkynyl;

[0059] M is a connector connected to G.

[0060] Wherein, ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 an alkynylene or amine group;

[0061] Preferably, M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 an alkynylene or amine group;

[0062] Preferably, M is wherein ring A is selected from M1 is selected from a single bond and C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 an alkynylene or amine group;

[0063] Preferably, M is selected from

[0064] Preferably, M is selected from

[0065] Preferably, M is selected from

[0066] Preferably, M is selected from

[0067] L is a linker between the linkers M and E, and L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) r OCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu- Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val- Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly),

[0068] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20;

[0069] Preferably, L is selected from the group consisting of one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is an integer selected from 1 to 20;

[0070] Preferably, L is selected from the group consisting of one or more of the following:

[0071] Preferably, L is selected from the following structures:

[0072] Preferably, L is selected from the following structures:

[0073] Preferably, L is selected from the following structures:

[0074] Preferably, L is selected from the following structures:

[0075] E is a structural fragment connecting L and D, wherein E is a single bond, -NHCH2- or selected from the following structures:

[0076] Preferably, E is a single bond, -NHCH2-,

[0077] Preferably, E is -NHCH2- or

[0078] Preferably, E is -NHCH2-;

[0079] Preferably, E is a single bond;

[0080] Preferably, E is

[0081] D is a cytotoxic drug fragment, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor; preferably, the microtubule inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof;

[0082] Preferably, the cytotoxic drug is selected from the compounds represented by Formula I or Formula II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds represented by Formula I or Formula II:

[0083] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0084] R3 is selected from H and -CO-CH2OH;

[0085] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0086] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0087] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0088] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl;

[0089] Preferably, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0090] The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug;

[0091] Preferably, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0092] and / or

[0093] x is selected from 1 to 10;

[0094] Preferably, Selected from the following structures:

[0095] Preferably, Selected from the following structures:

[0096] In another aspect, the present invention provides a drug-linker having the formula GM-[LED] x The structure shown, x is selected from 1 to 10, wherein:

[0097] GM is G is a leaving group for a nucleophilic substitution reaction (e.g.,

[0098] Halogen, methylsulfonyl, fluorophenol or ), or is hydroxyl (-OH), thiol (-SH) or amino (-NH2); or, G forms an unsaturated double bond with the adjacent atoms on ring A; Ring A is a 5-6 membered alicyclic heterocyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted with one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C2-20 Alkenylene, C 2- 20 an alkynylene or amine group;

[0099] The L, E and D structures are as defined above.

[0100] In some embodiments, GM is G is a methylsulfonyl group, or G forms a carbon-carbon double bond with the adjacent atoms on ring A; Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring through a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-10 Alkynylene or amino.

[0101] In some embodiments, GM is Selected from M1 is selected from a single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkynylene or amino.

[0102] In some embodiments, GM is

[0103] In some embodiments, GM is selected from

[0104] In some embodiments, GM is selected from

[0105] In some embodiments, x is selected from 1 to 10.

[0106] In some embodiments, the drug linker compound is selected from the following A-01 to A-34, B-01 to B-07, and C-01 to C-28:

[0107] In some embodiments, the drug linker compound of the present invention is selected from:

[0108] In some embodiments, the drug linker compounds described above may be optionally substituted with one or more suitable substituents.

[0109] In some embodiments, the drug linker compound described above has the following structure:

[0110] R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 5-12 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl, -C 1-6 Alkyl-C 6-10 Aryl and -C 1-6 Alkyl-5-12 membered heteroaryl; said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more selected from hydroxy, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 6-10 substituted by a substituent of an aryl group or a 5-12 membered heteroaryl group;

[0111] R 13 and R 14 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and 4-6 membered heterocyclic group; the alkyl, cycloalkyl and heterocyclic group are optionally substituted by one or more selected from hydroxyl, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 6- 10 substituted by a substituent of an aryl group or a 5-12 membered heteroaryl group;

[0112] R 15 Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, halogenated C 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 2-6 Alkenyl, C 2- 6-membered alkynyl and 3-6-membered heterocycloalkyl; R 16 H; or, R 15 、R 16and the atoms to which they are connected together form a 4-7 membered ring; the 4-7 membered ring is optionally substituted by one or more selected from hydroxyl, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 6-10 aryl, or a 5- to 12-membered heteroaryl substituent.

[0113] In some embodiments, R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl and indolyl-C 1-6 alkyl;

[0114] R 13 and R 14 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl; and

[0115] R 15 Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, halogenated C 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 2-6 Alkenyl, C 2- 6-membered alkynyl and 3-6-membered heterocycloalkyl; R 16 H; or, R 15 、R 16 and the atoms to which they are connected together form a 4-7 membered ring.

[0116] In some embodiments, R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-4 Alkyl, C 3-6 cycloalkyl, phenyl, benzyl, p-hydroxybenzyl and indolylmethyl.

[0117] In some embodiments, R 13 and R 14 are independently selected from hydrogen, C 1-4 Alkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclic groups.

[0118] In some embodiments, R 13 and R 14Each is independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and 4-6 membered heterocyclyl.

[0119] In some embodiments, R 15 Selected from hydrogen, C 1-4 Alkyl, C 3-6 Cycloalkyl, halogenated C 1-4 Alkyl, C 1-4 Alkyl-OC 1-4 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl and 3-6 membered heterocycloalkyl; or, R 15 、R 16 and the atoms to which they are attached together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.

[0120] In some embodiments, R 15 Selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1-4 Alkyl, C 1-4 Alkyl-OC 1-4 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl and 3-6 membered heterocycloalkyl; or, R 15 、R 16 and the atoms to which they are attached together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.

[0121] In some embodiments, R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-4 Alkyl, C 3-6 cycloalkyl, phenyl, benzyl, p-hydroxybenzyl, and indolylmethyl;

[0122] R 13 and R 14 each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 4-6 membered heterocyclyl;

[0123] R 15 Selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1-4 Alkyl, C 1-4 Alkyl-OC 1-4 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl and 3-6 membered heterocycloalkyl; or, R15 、R 16 and the atoms to which they are attached together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.

[0124] In another aspect, the present invention provides an intermediate compound having the following structure:

[0125] wherein X is selected from benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, nitrobenzenesulfonyl, benzoyl, pivaloyl, trityl, 4-methoxyphenyldiphenylmethyl, dimethoxytrityl, 2,4-dimethoxybenzyl, p-methoxybenzyl, and benzyl; a is an integer from 1 to 10, preferably an integer from 3 to 8; and R1, R2, and D are as defined above.

[0126] In some embodiments, X is selected from fluorenylmethoxycarbonyl (Fmoc).

[0127] In some embodiments, D is selected from:

[0128] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0129] R3 is selected from H and -CO-CH2OH;

[0130] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0131] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0132] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0133] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0134] In another aspect, the present invention provides an intermediate compound having the following structure:

[0135] In another aspect, the present invention provides an antibody drug conjugate having the formula Ab-[MLED] x In the structure shown, M, L, E, D, and x are as defined above, and Ab is an antibody or an antigen-binding fragment thereof that specifically binds to an antigen.

[0136] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, and ADC C-01 to ADC C-28 shown below: The definition of Ab in the following diagram is as described above, wherein the thiol group on the antibody and the drug linker compound form a thioether bond through an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate, and x represents the amount of drug loading:

[0137] in, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0138] Composition

[0139] In another aspect, the present application provides compositions that may comprise a plurality of ADCs as described herein. Each antibody molecule in the composition may be coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds of the invention. Thus, the composition is characterized by a "drug-antibody ratio" (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

[0140] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the antibody drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ~6.0,3.5~6.5,3.5~7.0,3.5~7.5,3.5~8.0,4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5 .5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0141] In some embodiments, the DAR value of the antibody drug conjugate is 4 to 8. It will be understood by those skilled in the art that the antibody drug conjugate described in this application can be prepared modularly. For example, first obtain the free form of the "drug-linker" (which can be understood as GM-[LED] x , wherein GM is the structural form before covalently linking to the antibody or antigen-binding fragment thereof), which is then covalently linked to the antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. Accordingly, the GM in the free form of the "drug-linker" is linked to one or more sulfhydryl (-SH), amino (-NH2), or carboxyl (-COOH) groups on the antibody or antigen-binding fragment thereof via a substitution reaction (e.g., removal of structures such as -SO2Me or -Br) or an addition reaction, where x is selected from 1 to 10.

[0142] Pharmaceutical composition

[0143] Pharmaceutical compositions of antibody drug conjugates and / or drug-linkers

[0144] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described in any one of the foregoing items or optionally the drug-linker described in any one of the foregoing items, and one or more pharmaceutical excipients.

[0145] The antibody drug conjugates described herein are generally formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same can be administered by any route appropriate to the subject to be treated.

[0146] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0147] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain embodiments, the additional pharmaceutically active agent is selected from a B7-H3 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic agent, or any combination thereof.

[0148] application

[0149] 1. Therapeutic uses of antibody-drug conjugates and / or drug-linkers

[0150] The antibody-drug conjugates, drug-linkers, or pharmaceutical compositions described herein can be used to treat a variety of diseases or conditions, such as B7-H3-positive tumors and Her2-positive tumors.

[0151] Therefore, the present application provides the use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and / or treating and / or assisting in the treatment of positive tumors.

[0152] The present application provides the use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and / or treating and / or adjuvant treatment of B7-H3-positive tumors.

[0153] The present application provides the use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and / or treating and / or adjuvant treatment of HER2-positive tumors.

[0154] At the same time, the present application also provides a method for preventing and / or treating and / or adjuvant treating positive tumors, which comprises the step of administering an effective amount of any of the above-mentioned antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same to a subject in need thereof.

[0155] The present application also provides a method for preventing and / or treating and / or adjuvant treating B7-H3 positive tumors, comprising the step of administering to a subject in need thereof an effective amount of any of the above-described antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same.

[0156] The present application also provides a method for preventing and / or treating and / or adjuvant treating HER2-positive tumors, comprising the step of administering an effective amount of any of the above-described antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same to a subject in need thereof.

[0157] The present application also provides use of any of the above-mentioned antibody-drug conjugates, drug linkers, or pharmaceutical compositions in inhibiting the proliferation of B7-H3-positive tumor cells.

[0158] The present application also provides use of any of the above-mentioned antibody-drug conjugates, drug linkers or pharmaceutical compositions in inhibiting the proliferation of Her2-positive tumor cells.

[0159] In certain embodiments, the antibody drug conjugate, drug linker or pharmaceutical composition is administered to cells in vitro or in vivo in a subject; for example, it is applied to a subject to inhibit the proliferation of tumor cells in the subject; or, it is applied to in vitro tumor cells (e.g., cell lines or cells from a subject) to inhibit the proliferation of tumor cells in vitro.

[0160] In the present application, B7-H3-positive tumors include solid tumors or hematological malignancies, such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), renal cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, and meningioma.

[0161] In the present application, the subject is preferably a mammal, such as bovine, equine, porcine, canine, feline, rodent, or primate; for example, a human.

[0162] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy can be applied simultaneously, separately, or sequentially with the above method.

[0163] In certain embodiments, the tumor involved in the pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

[0164] 2. Use of drug-linkers in the preparation of conjugates

[0165] The drug linker described above is used to prepare a conjugate, which includes an antibody-drug conjugate.

[0166] 3. Use of intermediate compounds in the preparation of drug linker compounds

[0167] The intermediate compound described above in the present invention is used to prepare a drug linker compound.

[0168] Preparation method

[0169] The present invention also provides a method for preparing the drug linker compound, which comprises the step of deprotecting the intermediate compound.

[0170] Method 1

[0171] In some embodiments, the method 1 includes a method for preparing compound A-05a, which includes the step of coupling with a compound of formula I;

[0172] A-05a:

[0173] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0174] R3 is selected from H and -CO-CH2OH;

[0175] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0176] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0177] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0178] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0179] In some embodiments, R1 and R2 are each independently selected from C 1-4 Alkyl, F, Cl, Br and I.

[0180] In some embodiments, R1 is selected from halogen, methyl, ethyl, propyl, and butyl, such as methyl and Cl.

[0181] In some embodiments, R2 is selected from F, Cl, Br, and I, such as F and Cl.

[0182] In some embodiments, R1 is selected from methyl and Cl, and R2 is selected from F and Cl.

[0183] Method 2

[0184] In some embodiments, the second method includes a method for preparing compound A-14-a, which includes the step of deprotecting IM-5-a to obtain a compound of formula IM-6-a;

[0185] Wherein, X is as defined in the intermediate compound described above, and R1 and R2 are as defined in Method 1.

[0186] In some embodiments, the deprotection reaction is as follows: a solvent is selected from N,N-dimethylformamide, and an alkylamine compound, such as diethylamine, is added.

[0187] In some embodiments, the deprotection is carried out at room temperature, and the reaction time is 1-5 h, such as 1-3 h.

[0188] In some embodiments, the second method further comprises the step of reacting IM-6-a with IM-2 to obtain compound A-14-a.

[0189] In some embodiments, the solvent for the reaction is selected from N,N-dimethylformamide and N,N-dimethylacetamide.

[0190] In some embodiments, the reaction further comprises the step of adding N,N-diisopropylethylamine.

[0191] Method 3

[0192] In some embodiments, the method three includes a method for preparing compound B-02-a, which includes the step of deprotecting B-02-4a;

[0193] wherein X is as described above in the intermediate compound, and R1, R2 and R3 are as described above in the compound of formula 1.

[0194] Method 4

[0195] In some embodiments, the method four includes a method for preparing compound C-07-a, which includes the steps of reacting C-07-6 with C-07-8a;

[0196] C-07-a:

[0197] Wherein, R1 and R2 are as defined in Method 1.

[0198] In some embodiments, C-07-8a in the fourth method is prepared by deprotection of C-07-7a;

[0199] C-07-7a: wherein X is as defined in the intermediate compound, and R1 and R2 are as defined in Method 1.

[0200] Method 5

[0201] In some embodiments, the method five includes a method for preparing compound C-10a, comprising the steps of reacting C-10-5a with C-07-8a;

[0202] C-10-a:

[0203] wherein R1, R2 and a are as defined above.

[0204] In some embodiments, C-10-5a in the fifth method is prepared by deprotection of C-10-4a;

[0205] In some embodiments, C-10-4a in the method five is prepared by reacting C-10-3 with compound A;

[0206] Method 6

[0207] In some embodiments, the method six includes a method for preparing compound C-17a, which includes the steps of reacting C-17-3a with C-07-8a;

[0208] wherein R1, R2 and a are as defined above; preferably, R1 is methyl, R2 is Cl and a is 3 or 8; R X is selected from CH or N.

[0209] In some embodiments, C-17-3a in the sixth method is prepared by oxidation of C-17-2a;

[0210] In some embodiments, C-17-2a in the sixth method is prepared by deprotection of C-17-1a;

[0211] In some embodiments, C-17-1a in the method six is ​​prepared by coupling reaction of C-10-2 or C-19-3 with compound A.

[0212] definition

[0213] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0214] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL 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 variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0215] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0216] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0217] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0218] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0219] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0220] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0221] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0222] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention 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. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0223] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0224] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0225] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0226] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0227] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0228] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0229] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0230] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0231] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC) of the interaction. 50 )express.

[0232] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant, KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473). KD, kon, and kdis values ​​can be measured using any valid method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Surface plasmon resonance techniques (e.g., Biacore) or Kinexa can also be used to measure the dissociation constant.

[0233] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0234] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0235] The terms "comprises," "comprising," "having," "containing," or "involving," and other similar forms thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0236] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20Alkyl", "C 1- 10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0237] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-20 Alkylene", "C 1-10 Alkylene", "C 3-10 Alkylene", "C 5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0238] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8 Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0239] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond and losing two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0240] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one (e.g., 1, 2, or 3) ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0241] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetrazine, and the like.

[0242] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a 5-6 membered aromatic heterocycle, e.g., a 5-6 membered nitrogen-containing aromatic heterocycle, e.g., a pyrimidine ring, etc.); two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.); the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), e.g., a 5-20 membered aromatic ring system.

[0243] As used herein, the term "suitable substituent" refers to modifications that can be made to a compound by one skilled in the art according to the needs of the compound substituent. "Suitable substituent" includes oxy (=O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 、R 9 Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, C 1- 6 alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0244] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0245] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0246] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or is at risk of suffering from the above-mentioned disease.

[0247] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0248] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0249] The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphoblastic leukemia. Hematological malignancies also include myeloma (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers. DETAILED DESCRIPTION

[0250] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0251] The information of the sequences involved in the present invention is described in the following table:

[0252] The abbreviations used in this document have the following meanings:

[0253] Abbreviation meaning

[0254] CDR Complementarity determining region in immunoglobulin variable region

[0255] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0256] VH antibody heavy chain variable region

[0257] VL Antibody light chain variable region

[0258] IgG immunoglobulin G

[0259] IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the Immunofluorescence Immunofluorescence (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0260] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0261] Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying CDR region boundaries based on the positions of structural loop regions (see, e.g., Chothia & Lesk (1987) J.

[0262] Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0263] The AbM CDR definition method is derived from the relevant research of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0264] mAb monoclonal antibody

[0265] EC 50 Concentration that produces 50% efficacy or binding

[0266] IC 50 Concentration that produces 50% inhibition

[0267] ELISA enzyme-linked immunosorbent assay

[0268] PCR polymerase chain reaction

[0269] HRP horseradish peroxidase

[0270] K D Equilibrium dissociation constant

[0271] Ka binding rate constant

[0272] Kd dissociation rate constant

[0273] ADCC Antibody-dependent cell-mediated cytotoxicity

[0274] CDC Complement-dependent cytotoxicity

[0275] FACS flow cytometry technology

[0276] CDR-H1 Complementarity-determining region 1 in the immunoglobulin heavy chain variable region

[0277] CDR-H2 Complementarity-determining region 2 in the immunoglobulin heavy chain variable region

[0278] CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region

[0279] CDR-L1 Complementarity-determining region 1 in the immunoglobulin light chain variable region

[0280] CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region

[0281] CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region

[0282] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0283] Nuclear magnetic resonance (NMR) 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0284] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0285] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0286] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0287] Example 1 N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (M-01)

[0288] Compound IM-1 (0.40 g, 640.59 μmol, its synthesis is described in patent CN 111936169A) and isotecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL). HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added and reacted at 25°C for 4 hours. DIPEA was removed under reduced pressure, and the mixture was freeze-dried with water to remove most of the DMF to obtain a crude product. The crude product was purified by preparative HPLC (under the following conditions) to obtain 273 mg of compound M-01.

[0289] Column: Waters XBridge Prep C18 OBD 45 mm × 450 mm × 8.0 μm

[0290] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0291] The structural characterization data of M-01 are as follows:

[0292] ESI-MS (m / z): 1034.4 [M+H] + .

[0293] Example 2: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-05)

[0294] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetrahydro-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 mL), the temperature was raised to 35 ° C, and the reaction was carried out for 16 hours. Then, (1S, 9S)- 1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was cooled to 5-15°C with ice water, and DMTMM (0.98 g, 3.53 mmol) was added, followed by dropwise addition of DIPEA (1.14 g, 8.84 mmol). The mixture was reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 mL), and concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to obtain 0.98 g of compound A-05.

[0295] A-05 separation and purification method is as follows:

[0296] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0297] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0298] A-05 structural characterization data are as follows:

[0299] MS m / z(ESI):1107.3[M+H]+

[0300] 1H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.3 1(m,1H),8.21-8.19(m,1H),8.17-8.09(m,2H),8.08-8.04(m,1H),7.30(s,1H),7.26 -7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H), 4.64(d,J=6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41( s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2.83-2.72(m,1H),2.58-2.56(m,2H),2.48 (s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J=7.2Hz,3H).

[0301] Example 3 N-((S)-10-benzyl-1-(((1S,9S)-5-fluoro-9-ethyl-9-hydroxy-4-chloro-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-07)

[0302] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetrahydro-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL), and the mixture was heated to 35° C. and reacted for 16 hours. Then, (1 S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione trifluoroacetate (30.0 mg, 0.053 mmol), HATU (30 mg, 0.079 mmol) and DIPEA (27.2 mg, 0.21 mmol) were reacted at 25°C for 16 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 26.4 mg of compound A-07.

[0303] The A-07 separation and purification method is as follows:

[0304] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0305] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0306] A-07 structural characterization data are as follows:

[0307] ESI-MS (m / z): 1111.3 [M+H] + .

[0308] Example 4 N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetrahydro-3-oxo-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-14)

[0309] Step 1:

[0310] Compound IM-4 (657 mg, 1.22 mmol) and compound 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL). HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) were then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 700 mg of compound IM-5.

[0311] The preparation method is as follows:

[0312] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0313] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0314] Step 2:

[0315] Compound IM-5 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 307 mg of compound IM-6.

[0316] The preparation method is as follows:

[0317] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0318] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0319] Step 3:

[0320] IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-14.

[0321] Its structural characterization data are as follows:

[0322] MS m / z(ESI):1002.4[M+H]+

[0323] The preparation method is as follows:

[0324] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0325] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0326] 1 H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2H z,1H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H ),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.5 6(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J=7.2Hz,3H).

[0327] Example 5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0328] Step 1:

[0329] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 30.00 mg of compound B-01-2.

[0330] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0331] Mobile phase A: acetonitrile; mobile phase B: water

[0332] Step 2:

[0333] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction mixture and allowed to react at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows) to obtain 20.00 mg of the trifluoroacetic acid salt of compound B-01.

[0334] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0335] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0336] The structural characterization data are as follows: ESI-MS (m / z): 1631.7 [M+H] + ,816.0[M / 2+H] + .

[0337] Example 6 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-02)

[0338] Step 1:

[0339] At 25 ° C, the methanesulfonate of 1-1 (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (58.74 mg, 112.88 μmol), N,N-diisopropylethylamine (43.76 mg, 338.63 μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 μmol) were added sequentially. The reaction was maintained at 25 ° C for 1 hour and the reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, water was added to the reaction solution and extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was separated by thin layer chromatography (dichloromethane: methanol = 15:1) to obtain 27.00 mg of compound B-02-1.

[0340] Step 2:

[0341] At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), and a dichloromethane solution (0.5 mL) of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol) was added in sequence. mL), maintained at 0 ° C for 0.5 hours; after replacing the residual triphosgene with nitrogen, (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-3,9-diazapentatriacontamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (43.46 mg, 40.99 μmol) in dichloromethane (1 mL) was added dropwise, and the reaction was maintained at 0 ° C for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by thin layer chromatography (dichloromethane: methanol = 15:1) to obtain 30.00 mg of compound B-02-2.

[0342] Step 3:

[0343] At 25°C, B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (62.98 mg, 206.26 μmol) and cuprous bromide (39.45 mg, 275.01 μmol) were added. The reaction was maintained at 25°C for 1 hour; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the prepared solution was lyophilized to obtain 150.00 mg of B-02-3 compound.

[0344] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0345] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0346] Step 4:

[0347] At 25 ° C, B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL), and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / glacial acetic acid (v / v = 13 / 1) (50 uL) was added dropwise. The reaction was maintained at 25 ° C for 0.5 hours and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the prepared solution was lyophilized to obtain 50.00 mg of B-02-4 compound.

[0348] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0349] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0350] Step 5:

[0351] At 25°C, B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (60.49 mg, 530.49 μmol) was added, and the reaction was maintained at 25°C for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high-performance liquid chromatography (conditions as follows), and the preparative solution was lyophilized to obtain 23.69 mg of B-02 compound.

[0352] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0353] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0354] The structural characterization data of B-02 are as follows:

[0355] ESI-MS (m / z): 1613.6 [M+H] + .

[0356] Example 7 N-((7S,10S,13S)-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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacarbonyl-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (C-07)

[0357] Step 1:

[0358] The raw material C-07-1 (4.80 g, 16.33 mmol), tributyl (2-methylsulfonylpyrimidin-4-yl) tin (16.27 g, 39.19 mmol) and bistriphenylphosphine palladium dichloride (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL). The reaction system was stirred at 110 ° C. under a nitrogen atmosphere for 5 hours. The reaction was monitored by LC-MS, and the reaction system was concentrated and purified by column chromatography (EA / PE = 0-50%) to obtain 1.36 g of C-07-2 compound.

[0359] Step 2:

[0360] Compound C-07-2 (510 mg, 1.33 mol) and NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and H₂O (2.5 mL). The reaction was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl, resulting in the precipitation of a large amount of solid. The filter cake was collected by filtration and dried to afford 380 mg of compound C-07-3.

[0361] Step 3:

[0362] Compound C-07-3 (315 mg, 850.32 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (246.31 mg, 935.35 μmol), HATU (484.99 mg, 1.28 mmol), and DIPEA (329.69 mg, 2.55 mmol) were added to DMF (3 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to yield 40 mg of compound C-07-3.

[0363] The preparation method is as follows:

[0364] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0365] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0366] Step 4:

[0367] Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL) and TFA (1.5 mL), reacted at 25° C. for 1.5 hours, monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 36 mg of compound C-07-5.

[0368] Step 5:

[0369] Compound C-07-5 (26 mg, 46.46 μmol), sodium periodate (99.37 mg, 464.57 μmol), and RuCl3·H2O (9.64 mg, 46.46 μmol) were dissolved in ACN (15 mL) and water (7.5 mL), and the mixture was reacted at 25°C for 40 minutes. The reaction was monitored by LC-MS, and water and ethyl acetate were added for extraction. The ethyl acetate layer was concentrated to obtain 28 mg of compound C-07-6.

[0370] Step 6:

[0371] Isotecan mesylate (600 mg, 1.13 mmol), (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxy-2,15-dioxy-4,7,10,13-tetraazaheptane-17-oic acid (IM-4, 610.17 mg, 1.13 mmol), HATU (643.81 mg, 1.69 mmol), and DIPEA (437.65 mg, 3.39 mmol) were added to DMF (6 mL) and reacted at 25°C for 16 h. The reaction was monitored by LC-MS. Water was added to the reaction solution, resulting in the precipitation of a large amount of solid. The solid was collected by filtration, dissolved in DCM, and concentrated to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 0-10%) to afford 660 mg of compound C-07-7.

[0372] Step 7:

[0373] Compound C-07-7 (660 mg, 688.94 μmmol) was dissolved in N,N-dimethylformamide (6 mL), and diethylamine (251.94 mg, 3.44 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 325 mg of compound C-07-8.

[0374] The preparation method is as follows:

[0375] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0376] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0377] Step 8:

[0378] Compound C-07-6 (15.95 mg, 25.58 μmol), C-07-8 (20 mg, 25.58 μmol), HATU (14.59 mg, 38.37 μmol), and DIPEA (9.92 mg, 76.75 μmol) were added to DMF (3 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to yield 7 mg of compound C-07.

[0379] Its structural characterization data are as follows:

[0380] ESI-MS (m / z): 1342.4 [M+H] + .

[0381] The preparation method is as follows:

[0382] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0383] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0384] Example 8 N-((7S,10S,13S)-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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-10)

[0385] Step 1:

[0386] The raw material C-10-1 (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol) XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and H2O (4 mL). The reaction system was stirred at 90°C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through celite, and water and ethyl acetate were added to the filtrate. The mixture was extracted and concentrated to obtain a crude product, which was purified by column chromatography (EA / PE = 0-25%) to obtain 710 mg of C-10-1 compound.

[0387] Step 2:

[0388] Compound C-10-1 (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and H2O (2 mL). The reaction was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1N HCl. A large amount of solid precipitated. The filter cake was collected by filtration and dried to obtain 560 mg of compound C-10-2.

[0389] Step 3:

[0390] Compound C-10-2 (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL), and m-CPBA (2.46 g, 12.1 mmol, 85% purity) was added to the reaction system. The reaction was allowed to proceed at 25°C for 12 hours, monitored by LC-MS. The solvent was dried under a stream of nitrogen to obtain a crude product, which was purified by preparative HPLC and freeze-dried to afford 153 mg of compound C-10-3.

[0391] The preparation method is as follows:

[0392] Chromatographic column: Phenomenex Luna C18 200*40mm*10um.

[0393] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid)

[0394] Mobile phase: [water (HCl)-ACN]; B%: 13%-43%, 10 min).

[0395] Step 4:

[0396] Compound C-10-3 (140 mg, 322.25 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (84.86 mg, 322.25 μmol), HATU (183.80 mg, 483.37 μmol), and DIPEA (124.94 mg, 966.75 μmol) were added to DMF (4 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to obtain 51 mg of compound C-10-4.

[0397] The preparation method is as follows:

[0398] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0399] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0400] Step 5:

[0401] Compound C-10-4 (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL), reacted at 25°C for 1 h, monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 45 mg of compound C-10-5.

[0402] Step 6:

[0403] Compound C-10-5 (31.91 mg, 51.17 μmol), C-07-8 (40 mg, 51.17 μmol), HATU (29.18 mg, 76.75 μmol), and DIPEA (19.84 mg, 153.50 μmol) were added to DMF (3 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to yield 13 mg of compound C-10.

[0404] Its structural characterization data are as follows:

[0405] ESI-MS (m / z): 1342.5 [M+H] +.

[0406] The preparation method is as follows:

[0407] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0408] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0409] Example 9: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-17)

[0410] Step 1:

[0411] C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (2.25 g, 8.10 mmol) were added to DMF (3 mL). HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added sequentially. The temperature was raised to 60°C and the reaction mixture was reacted for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude C-17-1 (3.8 g, 4.75 mmol), which was used directly in the next step without purification.

[0412] Step 2:

[0413] Dissolve C-17-1 (3.40 g, 5.40 mmol) in dichloromethane (30 mL) and add trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL). Stir the reaction mixture at 25°C for 2 hours. The reaction mixture is then concentrated, purified by preparative HPLC, and freeze-dried to afford C-17-2 (2.09 g, 3.64 mmol).

[0414] The preparation method is as follows:

[0415] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0416] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0417] Step 3:

[0418] C-17-2 (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), and then sodium periodate (208.79 mg, 976.15 μmol) and ruthenium trichloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system. The reaction was stirred at 25 ° C for 30 minutes. The reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction and concentrated to obtain C-17-3 (60 mg).

[0419] Step 4:

[0420] IM-6 (20 mg, 25.06 μmol), C-17-3 (16 mg, 25.06 μmol), HATU (19.05 mg, 50.11 μmol), and DIPEA (16.19 mg, 125.28 μmol) were added sequentially to DMF (3 mL). The reaction system was reacted at 25°C for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-17 (16 mg).

[0421] Its structural characterization data are as follows:

[0422] ESI-MS (m / z): 1371.4 [M+H] + .

[0423] The preparation method is as follows:

[0424] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0425] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0426] Example 10: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexadecane-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19)

[0427] Step 1:

[0428] C-19-1 (5.00 g, 16.9 mmol), (2-(methylthio)pyrimidin-5-yl)boronic acid (6.34 g, 37.3 mmol), XPhos Pd G3 (1.44 g, 1.70 mmol), and potassium phosphate (10.80 g, 50.9 mmol) were added to 1,4-dioxane (51.0 mL) and water (17.0 mL). The reaction system was purged with nitrogen three times and then reacted at 100°C for 5 hours. After the reaction system was cooled to room temperature, water (50.0 mL) was added to the reaction solution, which was filtered and concentrated to obtain the crude product. The product was slurried with petroleum ether and filtered again. The filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[0429] Step 2:

[0430] C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30.0 mL), MeOH (30.0 mL), and water (30.0 mL). LiOH·H2O (1.72 g, 40.9 mmol) was added and stirred at 25°C for 2 hours. The reaction mixture was adjusted to pH 3 with 1N aqueous hydrochloric acid. A solid precipitated and was filtered. The filter cake was dried under vacuum to afford C-19-3 (4.20 g).

[0431] Step 3:

[0432] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 3-(2-(2-aminoethoxy)ethoxyethoxyethyl)propionate (1.12 g, 4.04 mmol) were dissolved in DMF (20.0 mL). HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIPEA (2.09 g, 16.2 mmol) were added sequentially. The temperature was raised to 60°C and stirred for 2 hours. After the reaction system was cooled to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution. The aqueous phase was extracted twice with ethyl acetate (25.0 mL*2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude C-19-4 (2.50 g), which was used directly in the next step without purification.

[0433] Step 4:

[0434] C-19-4 (2.50 g, 3.96 mmol) was dissolved in dichloromethane (3.00 mL), and TFA (4.61 g, 40.4 mmol) was added. The reaction system was stirred at 25°C for 12 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain C-19-5 (1.20 g).

[0435] The preparation method is as follows:

[0436] Chromatographic column: Phenomenex luna C18 (150mm*25mm*10μm)

[0437] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0438] Step 5:

[0439] C-19-5 (1.10 g, 1.91 mmol) was dissolved in a mixed solvent of acetonitrile (30 mL) and water (15 mL), and ruthenium trichloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. The reaction system was reacted at 25 ° C for 1 hour, extracted with water (50 mL) and ethyl acetate (80 mL), and the organic phase was concentrated to obtain a crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain C-19-6 (130 mg).

[0440] Step 6:

[0441] IM-6 (20.0 mg, 0.025 mmol) and C-19-6 (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and stirred to dissolve. HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-19 (20.4 mg).

[0442] Its structural characterization data are as follows:

[0443] ESI-MS (m / z): 1372.4 [M+H] + .

[0444] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0445] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0446] Example 11: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazacyclotetradecane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21)

[0447] Step 1:

[0448] C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (4.03 g, 8.10 mmol) were added to DMF (40 mL). HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added sequentially. The reaction system was stirred at 60°C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford C-21-1 (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[0449] Step 2:

[0450] C-21-1 (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), and TFA (15.3 g, 134 mmol, 10 mL) was added. The reaction system was stirred at 25°C for 6 hours. Water (60 mL) and ethyl acetate (40 mL x 3) were added to the reaction solution, and the mixture was extracted. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by preparative HPLC and freeze-dried to obtain C-21-2 (2.93 g, 3.63 mmol).

[0451] The preparation method is as follows:

[0452] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0453] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0454] Step 3:

[0455] C-21-2 (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and then sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction system. The reaction was stirred at 25°C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain C-21-3 (155 mg).

[0456] Step 4:

[0457] IM-6 (27.91 mg, 34.97 μmol), C-21-3 (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol), and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL) and the reaction system was reacted at 25°C for 1 hour. The reaction solution was purified by HPLC and freeze-dried to obtain C-21 (15 mg).

[0458] Its structural characterization data are as follows:

[0459] ESI-MS (m / z): 1591.7 [M+H] + .

[0460] The preparation method is as follows:

[0461] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0462] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0463] Example 12: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazatetracan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23)

[0464] Step 1:

[0465] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (2.01 g, 4.04 mmol) were added to DMF (20.0 mL), followed by HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIEA (2.09 g, 16.2 mmol). The mixture was heated to 60°C and stirred for 2 hours. After cooling to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution for separation. The aqueous phase was extracted twice with ethyl acetate (25.0 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product C-23-1 (3.00 g), which was used directly in the next step.

[0466] Step 2:

[0467] C-23-1 (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), and TFA (15.4 g, 134 mmol) was added, followed by stirring at 25°C for 12 hours. The reaction mixture was concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to afford C-23-2 (1.20 g).

[0468] The preparation method is as follows:

[0469] Chromatographic column: Welch Ultimate C18 (150mm*25mm*5μm)

[0470] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0471] Step 3:

[0472] C-23-2 (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), and ruthenium trichloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol) were added. The system was reacted at 25 ° C for 1 hour, then extracted with water (10 ml) and ethyl acetate (40 ml). The organic phase was concentrated to obtain a crude product, which was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain C-23-3 (350 mg).

[0473] Step 4:

[0474] IM-6 (20.0 mg, 0.025 mmol) and C-23-3 (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL), and HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-23 (17.0 mg).

[0475] Its structural characterization data are as follows:

[0476] ESI-MS (m / z): 1592.6 [M+H] + .

[0477] The preparation method is as follows:

[0478] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0479] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0480] Example 13 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03)

[0481] Step 1: Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (B-03-1)

[0482] Dissolve (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) in DMF (50 mL). Add DIPEA (1.18 g, 9.12 mmol, 1.59 mL) dropwise. Add acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) dropwise with ice-cooling and stirring. Continue stirring for 1 hour. Add the reaction solution to 0.1 M dilute hydrochloric acid to precipitate a solid, which is then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0483] Its structural characterization data are as follows:

[0484] ESI-MS (m / z): 552.2 [M+1] + .

[0485] Step 2: Preparation of ethyl 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin-1-yl)amino)-2-oxoacetate (B-03-2)

[0486] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0 ° C under nitrogen protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise, and the reaction was stirred for 0.5 hour. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise and allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification on a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0487] Its structural characterization data are as follows:

[0488] ESI-MS (m / z): 1352.8 [M+1] + .

[0489] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-3)

[0490] 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,1 Ethyl 2-oxoacetate (200 mg, 0.122 mmol) (0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino) was dissolved in THF (3 mL) and MeOH (3 mL). A 1 mL aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise with stirring. Stirring was continued for 1 hour after the addition was complete. Dilute hydrochloric acid was added dropwise to the reaction mixture to neutralize the reaction. After concentration under reduced pressure, the mixture was directly transferred to the next step.

[0491] Its structural characterization data are as follows:

[0492] ESI-MS (m / z): 1596.7 [M+1] + .

[0493] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-4)

[0494] 4-((S)-35-Azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and the organic phase was concentrated to obtain a crude product, which was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0495] Its structural characterization data are as follows:

[0496] ESI-MS (m / z): 1323.6 [M+1] + .

[0497] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03)

[0498] 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ [3',4':6,7] indolizino[1,2-b]quinolin-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL). Cuprous bromide (9.42 mg, 0.066 mmol) was added and stirring continued for 2 hours. The reaction mixture was directly filtered and the concentrated crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0499] Its structural characterization data are as follows:

[0500] ESI-MS (m / z): 1628.7 [M+1] + .

[0501] The preparative high performance liquid chromatography method is as follows:

[0502] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0503] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0504] 2. Antibody Preparation

[0505] Fully humanized mice were immunized with human B7-H3-4Ig-His protein, and serum titers were monitored by ELISA and flow cytometry. The optimal mice were selected based on the titer results, and spleen cells were fused, screened, and subcloned. The binding activities of different monoclonal clones to human and monkey proteins and cells were tested, and the preferred clone 20G11G6 / 2# was obtained. The antibody sequence was modified by removing PTM sites, reducing PI, and removing ADCC activity in the heavy chain constant region. Finally, the fully human antibody 2#8890 (heavy chain variable region, SEQ ID NO: 3; light chain variable region, SEQ ID NO: 4) was obtained, along with the sequence of the human IgG1 heavy chain constant region with ADCC activity removed (SEQ ID NO: 31) and the human kappa light chain constant region (SEQ ID NO: 32), forming a complete humanized antibody (see Table 1). The antibody was codon-optimized and gene-synthesized by Nanjing GenScript Biotechnology Co., Ltd., and constructed into the pTT5 plasmid. The heavy and light chain plasmids were simultaneously transfected into CHO-S cells. Protein A purifies the expressed antibody in the supernatant to obtain the corresponding antibody protein 2#8890. The heavy chain amino acid sequence and light chain amino acid sequence of 2#8890 are shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0506] hIgG1 is an anti-chicken lysozyme antibody, the heavy chain variable region is fused to a mutant human IgG1 heavy chain constant region (SEQ ID NO: 31), and the light chain variable region is fused to a wild-type human kappa light chain constant region (SEQ ID NO: 32). The antibody hIgG1 was expressed and purified according to the above method.

[0507] The B7-H3 control antibody DS7300 was obtained from patent CN 103687945A. After codon optimization, the antibody heavy chain variable region nucleotide sequence was synthetically cloned into a human IgG1 heavy chain constant region containing a mutant (SEQ ID NO: 31), and the light chain variable region nucleotide sequence was synthesized into a pTT5 vector containing a wild-type kappa light chain constant region (SEQ ID NO: 32). The antibody DS7300 was expressed and purified as described above.

[0508] Table 1: Variable region and CDR amino acid sequences of 2#8890

[0509] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0510] The antibodies 2#8890, DS7300, and hIgG1 involved in the antibody-drug conjugates prepared in the following examples are the corresponding antibodies described in the second part above.

[0511] 1. Preparation of ADC 1 (DS7300-M-01, DAR 4)

[0512] Take 38.041 ml of homemade DS7300 antibody (26.287 mg / mL), adjust the pH to 7.4 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (3.413 mL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 4.096 mL, pH 7.4) solution in an ice bath, mix well, and let it stand at 4°C overnight. A 6-fold amount of M-01 (4.18 mL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed. The mixture was reacted at 4°C for 4 h. A cysteine ​​solution (10 mM, 6.826 mL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 6.826 mL) was added. After 30 minutes, a DHAA solution (10 mM, 6.826 mL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 1 (DS7300-M-01). The DAR value was determined by mass spectrometry to be 3.8.

[0513] 2. Preparation of ADC 2 (hIgG1-M-01, DAR 4)

[0514] Take 1.2245 ml of hIgG1 antibody (24.5 mg / mL), adjust the pH to 7.3 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (52.04 uL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.89 uL, pH 7.3) solution in an ice bath, mix well, and incubate at 4°C overnight. A 6-fold amount of 171 (127.44 μL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was reacted at 4°C for 4 h. A cysteine ​​solution (10 mM, 208.15 μL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 208.15 μL) was added. After 30 minutes, a DHAA solution (10 mM, 208.15 μL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 2 (i.e., hIgG1-M-01). The DAR value was determined by mass spectrometry to be 4.46.

[0515] 3. Preparation of ADC 3 (2#8890-M-01, DAR 4)

[0516] Take 9.36 ml of 2#8890 antibody (3.205 mg / mL), adjust the pH to 7.4 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (57.31 uL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.9 uL, pH 7.4) solution in an ice bath, mix well, and let it stand at 4°C overnight. A 6-fold amount of M-01 (124.9 μL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed. The mixture was allowed to react at 4°C for 4 h. A cysteine ​​solution (10 mM, 208.2 μL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 208.2 μL) was added. After 30 minutes, a DHAA solution (10 mM, 208.2 μL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC3 (2#8890-M-01). The DAR value was determined by mass spectrometry to be 3.89.

[0517] 4. Preparation of ADC 4 (hIgG1-A-05, DAR 8)

[0518] 0.943 ml of hIgG1 antibody (11 mg / mL) was diluted with 47 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 57 μL) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-05 dissolved in dimethyl sulfoxide (103 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 4 (hIgG1-A-05). The DAR value, determined by mass spectrometry, was 8.03.

[0519] 5. Preparation of ADC 5 (2#8890-A-05, DAR 8)

[0520] 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 153 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 114.5 μL, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-05 dissolved in dimethyl sulfoxide (219.1 μL, 10 mM) was then added and mixed thoroughly. The mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 5 (2#8890-A-05). The DAR value was 7.90 as determined by mass spectrometry.

[0521] 6. Preparation of ADC 6 (hIgG1-A-07, DAR 8)

[0522] 0.518 ml of hIgG1 antibody (19.3 mg / mL) was diluted with 25.9 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 38.1 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-07 dissolved in dimethyl sulfoxide (69.2 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 6 (hIgG1-A-07). The DAR value, determined by mass spectrometry, was 8.04.

[0523] 7. Preparation of ADC 7 (2#8890-A-07, DAR 8)

[0524] 1.017 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 50.85 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 38.2 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-07 dissolved in dimethyl sulfoxide (87.6 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 7 (2#8890-A-07). The DAR value, determined by mass spectrometry, was 7.76.

[0525] 8. Preparation of ADC 8 (hIgG1-A-14, DAR 8)

[0526] 1.9126 ml of hIgG1 antibody (18.3 mg / mL) was diluted with 95.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 66.86 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-14 dissolved in dimethyl sulfoxide (260.53 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. The buffer was then exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 8 (hIgG1-A-14). The DAR value, as determined by mass spectrometry, was 8.0.

[0527] 9. Preparation of ADC 9 (2#8890-A-14, DAR 8)

[0528] 3.6788 ml of 2#8890 antibody (10.873 mg / mL) was diluted with 183.94 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 152 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-14 dissolved in dimethyl sulfoxide (292.26 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 9 (2#8890-A-14). The DAR value was 7.22 as determined by mass spectrometry.

[0529] 10. Preparation of ADC 10 (hIgG1-B-01, DAR 8)

[0530] 1.533 ml of hIgG1 antibody (19.57 mg / mL) was diluted with 76.65 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 114.5 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-01 dissolved in dimethyl sulfoxide (212.4 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 10 (hIgG1-B-01). The DAR value, determined by mass spectrometry, was 8.03.

[0531] 11. Preparation of ADC 11 (2#8890-B-01, DAR 8)

[0532] 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 152.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 114.5 μL, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-01 dissolved in dimethyl sulfoxide (212.4 μL, 10 mM) was then added and mixed thoroughly. The mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 11 (2#8890-B-01). The DAR value was 7.75 as determined by mass spectrometry.

[0533] The ADC samples after coupling were subjected to LC-MS molecular weight analysis.

[0534] Chromatographic determination conditions:

[0535] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0536] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0537] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0538] Mass spectrometry conditions:

[0539] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0540] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; Accumulation time 0.5s;

[0541] m / z 600-4000; Time bins to sum 40.

[0542] 12. Preparation of ADC 12 (2#8890-C-07, DAR 4)

[0543] 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 9.5 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. An 8-fold amount of C-07 dissolved in dimethyl sulfoxide (14.6 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 12 (2#8890-C-07). The DAR value was 4.12 as determined by mass spectrometry.

[0544] 13. Preparation of ADC 13 (2#8890-C-10, DAR 4)

[0545] 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 9.5 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. An 8-fold amount of C-10 dissolved in dimethyl sulfoxide (14 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 13 (2#8890-C-10). The DAR value was 4.17 as determined by mass spectrometry.

[0546] The ADC samples after coupling were subjected to LC-MS molecular weight analysis.

[0547] Chromatographic determination conditions:

[0548] Liquid chromatography column: ACQUITY UPLC MAbPac BEH SEC;

[0549] Mobile phase A: 20 mM NH4Ac;

[0550] Flow rate: 0.1 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0551] Mass spectrometry conditions:

[0552] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0553] GS1 55; GS2 55; CUR 30; TEM 450; ISVF 5500; DP 75; CE 5; Accumulation time 0.5s; m / z 900-7000; Time bins to sum 40.

[0554] 14. Preparation of ADC 14 (2#8890-C-17, DAR 4)

[0555] 0.292 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 14.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 4.77 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5-fold amount of C-17 dissolved in dimethyl sulfoxide (8.76 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 14 (2#8890-C-17). The DAR value was 3.86 as determined by mass spectrometry.

[0556] 15. Preparation of ADC 15 (2#8890-C-19, DAR 4)

[0557] 0.584 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-19 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 15 (2#8890-C-19). The DAR value was 4.05 as determined by mass spectrometry.

[0558] 16. Preparation of ADC 16 (2#8890-C-21, DAR 4)

[0559] 0.584 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-21 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 16 (2#8890-C-21). The DAR value was 3.92 as determined by mass spectrometry.

[0560] 17. Preparation of ADC 17 (2#8890-B-03, DAR 8)

[0561] 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 93.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 112.65 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. An 11-fold amount of B-03 dissolved in dimethyl sulfoxide (227.58 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 17 (2#8890-B-03, DAR 8). The DAR value, determined by mass spectrometry, was 7.82.

[0562] 18. Preparation of ADC 18 (2#8890-B-03, DAR 8)

[0563] 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 58.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 112.65 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-03 dissolved in dimethyl sulfoxide (211.1 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 18 (2#8890-B-03, DAR 8). The DAR value, determined by mass spectrometry, was 7.23.

[0564] 19. Preparation of ADC 19 (19F6-B-02)

[0565] The antibody 19F6_Hu35v1 involved in the following examples is the 19F6_Hu35v1 antibody described in international patent application WO2022253035A1, and was prepared using the method described in Example 2 of the patent.

[0566] The sample was prepared by coupling as follows:

[0567] 0.46 ml of 19F6-Hu35v1 antibody (11.0 mg / mL) was diluted with 0.1 M edetate disodium solution (pH 7.7), then adjusted to pH 7.7 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, mixed, and allowed to stand at room temperature for 90 minutes. A 10-fold amount of B-02 dissolved in dimethyl sulfoxide was added to the solution, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). Sucrose and Tween 20 were then added and mixed to obtain the antibody-drug conjugate, ADC 19 (19F6-B-02). The DAR value was 7.92 as determined by mass spectrometry.

[0568] 20. ADC 20 (Trastuzumab-A-05, DAR 8)

[0569] 2.469 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 123 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 151.57 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-05 (290.09 μL, 10 mM) dissolved in dimethyl sulfoxide was then slowly added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-05). The DAR value was determined by mass spectrometry to be 8.04.

[0570] 21. ADC 21 (Trastuzumab-A-14, DAR 8)

[0571] 2.564 mL of trastuzumab antibody (15.6 mg / mL) was diluted with 128.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 151.57 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-14 dissolved in dimethyl sulfoxide (290.1 ​​μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-14). The DAR value was determined by mass spectrometry to be 8.02.

[0572] 22. ADC 22 (Trastuzumab-A-24, DAR 8)

[0573] 0.617 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 30.86 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 37.89 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 14-fold amount of A-24 dissolved in dimethyl sulfoxide (98.42 μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-24). The DAR value, determined by mass spectrometry, was 7.37.

[0574] 23. ADC23 (Trastuzumab-A-32, DAR 8)

[0575] 0.423 mL of trastuzumab antibody (4.73 mg / mL) was diluted with 21.17 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 7.58 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-32 dissolved in dimethyl sulfoxide (13.92 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-32). The DAR value, determined by mass spectrometry, was 6.77.

[0576] 24. ADC24 (Trastuzumab-C-21, DAR 4)

[0577] 0.617 mL of trastuzumab antibody (16.2 mg / mL) was diluted with 30.86 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 19.83 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 19.1 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 5.5-fold increase in the amount of C-21 dissolved in dimethyl sulfoxide (38.28 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-C-21). The DAR value determined by mass spectrometry was 4.41, with DAR4 accounting for 60.06%.

[0578] IV. Detecting the activity of antibody-drug conjugates

[0579] 1. Dynamic affinity detection of anti-human B7-H3 antibody conjugates

[0580] The dynamic affinity of the anti-human B7-H3 antibody conjugate to human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins was tested using ForteBio (Pall Life Sciences). The specific method is as follows: the test conjugate was diluted to 5 μg / ml in PBST (0.02% Tween-20), and human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins were gradiently diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The test conjugate was then captured using a Protein A Sensor (Pall Life Sciences) in PBST (0.02% Tween-20) solution for 60 s, then bound to each of the four proteins for 60 s, and then dissociated for 180 s. The measured results were opened in Data Analysis 11.0 software, and the 1:1 mode and global fitting were selected for analysis to obtain affinity constants. The results are shown in Table 2, which show that the conjugate prepared from the drug linker compound of the present invention binds to monkey B7-H3 but not to rat B7-H3.

[0581] Table 2: Dynamic affinity test results of anti-human B7-H3 antibody conjugates

[0582] 2. Cellular affinity testing of anti-human B7-H3 antibody conjugates

[0583] The affinity of the anti-human B7-H3 fully human antibody conjugate to human colon cancer cells HT29 (Cell Bank of the Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was tested using a flow cytometer (Beckman, Cytoflex). The affinity of the anti-human B7-H3 fully human antibody conjugate to CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig was tested. The cross-species affinity of the anti-human B7-H3 fully human antibody conjugate to CHOS-rat B7-H3 and CHOS-monkey B7-H3 was tested. Adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, counted, and the cell density adjusted to 4.0 × 10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension was added to each well of a 96-well pointed bottom plate (cell number 2×10 5 1% BSA was used to dilute the antibody conjugate (starting at a final concentration of 10 μg / ml, with a 3-fold serial dilution for a total of 11 concentration points). A hIgG1 antibody conjugate was used as a control (final concentration of 10 μg / ml). 50 μl of the diluted antibody was added to the conical bottom plate containing the cells and incubated at 4°C for 60 min. The cells were washed twice with 1% BSA, and 50 μl of the diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. The cells were washed twice with 1% BSA, resuspended in 200 μl of 1% BSA, and analyzed by flow cytometry. Data processing: Median PE values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC values. 50 .

[0584] Among the affinity results of anti-human B7-H3 antibody conjugates for HT29, NCI-N87, HCC1806, and HCC827 tumor cells, the EC value of ADC17 for HT29 tumor cells is 50 The EC value for HCC827 tumor cells is 3.213 ng / ml. 50The affinity results of the other antibody conjugates are shown in Table 3. The affinity results of the anti-human B7-H3 fully human antibody conjugate for CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2IG cells are shown in Table 4. The affinity results of the anti-human B7-H3 antibody conjugate for CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells are shown in Table 5. The results show that the conjugate prepared by the drug linker compound of the present invention binds to monkey B7-H3 overexpressing cells, but does not bind to rat B7-H3 overexpressing cells.

[0585] Table 3: ADC tumor cell affinity determination results

[0586] Table 4: Results of cell affinity determination of CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig

[0587] Table 5: Results of cell affinity assays for CHOS-rat B7-H3 and CHOS-monkey B7-H3

[0588] 3. Detection of cellular endocytic activity of anti-human B7-H3 antibody conjugates

[0589] The endocytic activity of the anti-human B7-H3 antibody conjugate in human gastric cancer cells NCI-N87 (ATCC), human breast squamous carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was detected using a flow cytometer (Thermo, model Attune NxT). Adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted. The cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4The 96-well plate was placed in a 37°C, CO2 constant temperature incubator and incubated for 24 h. Remove the 96-well plate, aspirate the medium, and add 50 μl of fresh complete medium to each well; dilute the test antibody conjugate with complete medium to a total of 6 concentration points; dilute 300 μg / ml pHrodo reagent (Thermo, Cat# Z25612) to 12 μg / ml in complete medium (final pHrodo concentration is 3 μg / ml); mix the serially diluted test antibody and diluted pHrodo reagent in a 1:1 ratio (30 μl:30 μl) and incubate at room temperature in the dark for 30 min; add 50 μl of the test antibody and pHrodo reagent mixture to the 96-well plate and incubate at 37°C, 5% CO2 for 24 h; remove the 96-well plate, aspirate the medium, wash once with sterile PBS, add 100 μl of Trypsin-EDTA (0.25%) to each well to digest the cells, and then add 100 μl of complete medium to neutralize; disperse the cells in the wells by pipetting and analyze them on a FACS machine. Data processing: Median YL-1H values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC 50 As shown in Table 6, the antibody drug conjugates (eg, ADC 5 and ADC 9) prepared using the drug linker compounds of the present invention all have good endocytic activity.

[0590] Table 6: ADC cell endocytosis activity assay results

[0591] 4. In vitro cell killing assay of anti-human B7-H3 antibody-drug conjugates

[0592] Adherent A375, Calu6-B7-H3, and U87MG-B7-H3 cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted. The cell density was adjusted to 1×10 4 , 5×10 4 , 1×10 4 Cells / ml, add 100 μl of cell suspension to each well of a 96-well plate (cell numbers are 1000, 5000, and 1000 / well, respectively), and place the 96-well plate in a 37°C, CO2 constant temperature incubator for 24 hours. Use complete culture medium to dilute the ADC to be tested, starting with a final concentration of 3333.3nM, and 4-fold serial dilution for a total of 12 concentration points; take 100 μl of diluted ADC and add it to a 96-well plate, and incubate at 37°C, 5% CO2 for 4 to 7 days. Remove the 96-well plate, add 20 μl of CCK8 reagent to each well, incubate at 37°C for 2 to 3 hours, and use a microplate reader to detect OD 450nm The signal values ​​were then imported into GraphPad Prism 6 software to calculate the IC 50, the results are shown in Table 7.

[0593] Table 7 ADC in vitro cell killing results

[0594] The results showed that the antibody-drug conjugate prepared by the drug linker compound of the present invention has a clear killing effect on tumor cells.

[0595] 5. In vivo efficacy testing of different conjugated drugs of 2#8890 in the HCC1806 model

[0596] Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right scapula with 2×10 6 HCC1806 cells were suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-200 mm 3 At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 6 groups of 6 mice based on tumor volume and body weight. They were given a single dose via tail vein injection (DAR4 group: 10 mg / kg, DAR8 group: 5 mg / kg). Tumor volume and body weight were measured twice a week after administration. Specific results are shown in Table 8.

[0597] Table 8 Analysis of the efficacy of different conjugated drugs of 2#8890 on HCC1806 cell tumor-bearing mouse model

[0598] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0599] Results showed that the weight of mice in the drug-treated groups remained stable and well-tolerated. Ninety-five days after a single dose, the tumors in the ADC 5 group of mice completely regressed. This demonstrates that the antibody-drug conjugates prepared using the drug-linker compounds of the present invention exhibit significant tumor-suppressing effects.

[0600] 6. Efficacy testing of different doses of antibody-drug conjugates in the HCC1806 model

[0601] Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right scapula with 2×106 HCC1806 cells were suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-200 mm 3 At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 12 groups of 6 mice each based on tumor volume and body weight. A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly after administration. Detailed results are shown in Table 9.

[0602] Table 9 Analysis of the efficacy of different doses of antibody-drug conjugates in the HCC1806 cell tumor-bearing mouse model

[0603] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0604] The results showed that both ADC 5 and ADC 9 groups had good efficacy, and the mice maintained stable body weight and had good tolerance, indicating that the antibody-drug conjugates prepared using the drug linker compounds of the present invention had significant tumor inhibitory effects and good safety.

[0605] 7. Efficacy testing of different antibody-drug conjugates in the NCI-N87 model

[0606] Human gastric cancer cells NCI-N87 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right shoulder blade with 5×10 6 NCI-N87 cells were suspended in 0.1 ml of PBS matrix gel. When the average tumor volume grew to about 100-200 mm 3 At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 8 groups of 6 mice each based on tumor volume and animal body weight. The drug was injected into the tail vein twice a week. Tumor volume and body weight were measured twice a week after administration. The specific results are shown in Table 10.

[0607] Table 10 Analysis of the efficacy of different antibody-drug conjugates in the NCI-N87 cell tumor-bearing mouse model

[0608] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0609] The results showed that the body weight of mice in each drug administration group was stable and well tolerated, indicating that the antibody-drug conjugate prepared by the drug linker compound of the present invention has a significant tumor inhibitory effect and good safety.

[0610] 8. Detection of Anti-Human B7-H3 Antibody-Drug Conjugate Binding Activity to Fc Receptors

[0611] ForteBio (Pall Life Sciences) was used to test the dynamic affinity of the 2#8890 antibody-drug conjugate for the human Fc receptor proteins CD16a, CD32a, CD32b, C1q, and FcRn. The specific method is as follows: The biotinylated proteins to be tested were captured using an SA Sensor (Pall Life Sciences) in PBST solution. The test antibody and conjugate were diluted to a starting concentration of 5000 nM in PBST and then diluted two-fold at seven concentration points. Binding and dissociation were performed, and the measured results were opened in Data Analysis 11.0 software using a 1:1 mode and global fitting. The results were analyzed to obtain association rates, dissociation rates, and affinity constants. The results are shown in Table 11.

[0612] Table 11 Detection of Fc receptor binding activity of antibodies and conjugates

[0613] The results show that the antibody-drug conjugates prepared from the drug linker compounds of the present invention do not bind to Fc receptors CD16a, CD32a, CD32b, and C1q proteins, can reduce Fc receptor-mediated nonspecific killing, and improve drug safety. At the same time, these conjugates retain FcRn protein binding activity and do not affect the half-life of the drug.

[0614] 9. Pharmacokinetic study of total antibody (Tab), ADC, and payload in serum of cynomolgus monkeys after multiple intravenous injections of anti-human B7-H3 antibody-drug conjugates

[0615] ADCs (ADC 5, ADC 9), total antibodies (TAb), and payload were quantitatively detected in cynomolgus macaque serum using ELISA and LC-MS / MS. The standard curves for both the ADCs (ADC 5, ADC 9) and total antibodies (TAb) assays had a quantification range of 11.72 to 3000 ng / mL, and the payload assay had a linear range of 0.1 to 40 ng / mL. Both the ADCs (ADC 5, ADC 9) and total antibodies (TAb) assays used B7-H3 protein as the capture protein, coated in a 96-well microtiter plate. Total antibodies (TAb) were then detected using goat anti-human IgG-HRP. For the ADCs (ADC 5, ADC 9), anti-toxin mouse antibodies and goat anti-mouse IgG were used as secondary and detection antibodies, respectively. Color development was achieved through the interaction of the enzyme and substrate, and the results were read on a SpectraMax i3x (Molecular Devices) microplate reader. The 4-P parameter method was used to fit a standard curve and calculate the concentration of each sample. ADC (ADC 5, ADC 9) and total antibody (TAb) concentrations were positively correlated with the color intensity. LC-MS / MS was performed on a Shimadzu LC 30-AD liquid chromatography system coupled to a SCIEX QTRAP 5500+ (SCIEX) mass spectrometer, using (+)ESI ionization and multiple reaction monitoring (MRM) mode. The column was an Xbridge C18 50*4.6mm, 5μm. The ion pairs for compounds 1-10 were 510.2 / 435.2. Sample pretreatment involved protein precipitation with acetonitrile. Results: After multiple intravenous injections of ADC5 and ADC9 in cynomolgus monkeys, test results showed that the ADC molecules prepared using the drug-linker compounds of the present application (e.g., ADC5 and ADC9) exhibited good pharmacokinetic properties, were relatively stable in the systemic circulation, and released little free toxin.

[0616] Table 12 Pharmacokinetic parameters of TAb in serum after the last intravenous injection of ADC at 50 mg / kg

[0617] Table 13 Pharmacokinetic parameters of ADC in serum after the last intravenous injection of 50 mg / kg

[0618] Table 14 Pharmacokinetic parameters of Payload in serum after the last intravenous injection of ADC at 50 mg / kg

[0619] 10. Repeated-dose toxicity test

[0620] The repeated-dose toxicity study included a repeated toxicity study in which ADC5 and ADC 9 were intravenously injected four times into cynomolgus monkeys.

[0621] This study included three groups, one animal per group of each sex, each receiving 30 mg / kg of ADC5 or ADC9 intravenously with saline (volume: 10 mL / kg) once weekly for two doses. The dose was then increased to 50 mg / kg of ADC5 or ADC9 with saline once weekly for two doses. No test article-related deaths or near-deaths were observed during the study. During the ADC5 group, animals exhibited decreased appetite, hair loss, skin pigmentation, and weight loss. Female monkeys showed decreased white blood cell count (WBC), neutrophil urea (NEUT), lysozyme (LYM), and mononuclear cell count (MONO). Male monkeys showed decreased red blood cell count (RBC), hematocrit (HGB), and hematocrit (HCT), and increased fibroblast growth factor (FGB), with a trend toward recovery during the recovery period. During the ADC9 group, animals exhibited decreased appetite. Female monkeys showed decreased WBC, neutrophil urea (NEUT), LYM, and hematocrit (MONO), and increased fibroblast growth factor (FBG). Male monkeys showed increased FBG and decreased RBC, HGB, and HCT. During the recovery period, hyperpigmentation and mild hair loss at the site of administration were also observed, with all other changes showing a trend toward recovery. In this study, cynomolgus monkeys received intravenous injections of 30 mg / kg of ADC 5 and ADC 9 once weekly for two consecutive weeks. The dose was then increased to 50 mg / kg of ADC 5 and ADC 9 once weekly for two consecutive weeks. All animals tolerated the treatment, with the highest no-serious-toxicity dose (HNSTD) being 50 mg / kg.

[0622] 11. Antibody-drug conjugates inhibit tumor growth in a mouse subcutaneous transplant tumor model

[0623] The preparation containing the ADC of the present invention was administered via tail vein injection to a CDX mouse model subcutaneously transplanted with human breast squamous cell carcinoma cell HCC1806. The tumor volume and animal body weight changes were measured twice a week to calculate the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice.

[0624] Experimental animals: Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd.)

[0625] Cell line: Human breast squamous carcinoma cell line HCC1806 (ATCC)

[0626] Experimental methods:

[0627] HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells were collected during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nude mice to establish a breast squamous cell carcinoma model. The average tumor volume was approximately 200 mm. 3Around 30 days, the mice were randomly divided into groups according to the size of the tumor and given drugs separately. The groups and their dosages were as follows: vehicle control group (i.e. negative control, Vehicle group): given 0.9% Nacl injection; ADC 5: dosage of 3 mg / kg; ADC 9: dosage of 3 mg / kg; ADC 11: dosage of 3.16 mg / kg; ADC 17: dosage of 3.32 mg / kg. Each group was injected with tail vein (iv) and given on Day 0, for a total of 1 dose. After administration, the body weight of the mice was measured twice a week, and the long and short diameters of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0628] The tumor growth inhibition rate (TGI) was calculated using the following formula: T末 >V T0 ,TGI(%)=[1-(V T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 ,TGI(%)=[1-(V T末 - V T0 ) / V T0 ]*100%.

[0629] Where V T末 : Mean tumor volume of treatment group at the end of the experiment

[0630] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0631] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0632] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0633] The relative tumor proliferation rate T / C (%) was calculated using the following formula: T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0634] The ADCs of the present invention demonstrated significant tumor growth inhibition in an HCC1806 breast squamous cell carcinoma xenograft model. On day 14, compared to the vehicle group, the tumor growth inhibition rates (TGI) of ADCs 5, 9, 11, and 17 were 96.68%, 98.50%, 82.61%, and 86.69%, respectively, demonstrating significant differences compared to the control group. During treatment, no animals in each treatment group experienced mortality or significant weight loss, and no significant drug toxicity was observed. The ADCs of the present invention were well tolerated by mice. Specific results are shown in Table 15.

[0635] Table 15 Analysis of the efficacy of different antibody-drug conjugates in HCC1806 cell tumor-bearing mouse models

[0636] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0637] 12. Inhibitory effect of antibody-drug conjugates on cell activity in vitro

[0638] (1) Cell plating: First, culture tumor cells NCI-H1975 and HT-29 in the corresponding culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to the appropriate concentration for plating. The tumor cell sources are shown in Table 16.

[0639] Table 16: Tumor cell sources

[0640] After the cells adhered, the culture medium in the cells was removed, and the diluted antibody-drug conjugate ADC 19 was added to the above plate wells and incubated for 96 hours.

[0641] In vitro cell activity assay: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes and then detect, read with microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM The background RLU was obtained from the culture wells without cells, and the control RLU was obtained from the culture wells with cells but without compound. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) × 100%. The half-maximal inhibitory concentration (IC) of ADC 19 was calculated according to the four-parameter model curve fitting. 50 ).

[0642] The half-maximal inhibitory concentration (IC50) of ADC 19 on NCI-H1975 cell line 50)5.00μg / mL; the half-maximal inhibitory concentration (IC) of ADC 19 on HT-29 cell line 50 )4.56μg / mL.

[0643] The results show that the antibody-drug conjugate (eg, ADC19) formed by the drug linker of the present invention has significant tumor cell killing activity.

[0644] 13. Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0645] NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO₂. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume reached approximately 160 mm³, the mice were randomly divided into the following groups based on tumor size: vehicle control (negative control, vehicle group), trastuzumab-A-05 1 mg / kg group, trastuzumab-A-14 1 mg / kg group, and trastuzumab-A-24 1 mg / kg group. Each group received the drug via tail vein (iv) injection on Day 0, Day 7, and Day 14, for a total of three doses. After administration, the body weight of mice was measured twice a week, and the long and short diameters of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b2, where a and b represent the long and short diameters of the tumor, respectively. The death of animals was observed and recorded every day.

[0646] The ADCs of the present invention demonstrated significant tumor growth inhibition in the NCI-N87 gastric cancer xenograft model. Compared with the vehicle control group, the tumor growth inhibition rates (TGI) of the 1 mg / kg trastuzumab-A-05, 1 mg / kg trastuzumab-A-14, and 1 mg / kg trastuzumab-A-24 groups were 81.62%, 107.38%, and 61.43%, respectively. On Day 30, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. During treatment, the mice tolerated the ADCs prepared using the drug-linker compounds of the present invention well. Specific results are shown in Table 17.

[0647] Table 17 Human gastric cancer cell NCI-N87 CDX model

[0648] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0649] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A drug linker compound having the formula GM-[LED] x The structure shown, wherein: G is a functional group or leaving group capable of reacting with specific amino acids or sugar groups; M is a linker connected to G, and the M is Wherein, ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkyne or amino, the C 1-20 Alkylene, C 2- 20 Alkenylene, C 2-20 The alkynylene or amine group is optionally substituted with one or more suitable substituents; L is a linker between the linkers M and E, and L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs, and short peptides composed of amino acids, Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20; E is a structural fragment connecting L and D, wherein E is a single bond, -NHCH2- or selected from the following structures: D is a cytotoxic drug fragment; and / or x is selected from 1 to 10.

2. The drug linker compound according to claim 1, wherein G is selected from halogen, halogenated C 1-6 Alkyl, C 1- 6-sulfonyl, halogenated C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 Sulfoxide, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Sulfonyl, halo C 1-6 Sulfonyl, halosulfonyl, C 1-6 Sulfonate group, halogenated C 1-6 Sulfonate group, C 1-6 Sulfinate group, C 1-6 The sulfoxide, alkenyl, alkynyl and alkynyl-containing structural fragments are optionally substituted with one or more suitable substituents.

3. The drug linker compound according to claim 1 or 2, wherein M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 Alkyne or amino, the C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

4. The drug linker compound according to any one of claims 1 to 3, wherein M is wherein ring A is selected from M1 is selected from a single bond and C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkyne or amino, the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 The alkynylene or amine group is optionally substituted with one or more suitable substituents.

5. The drug linker compound according to any one of claims 1 to 4, wherein M is selected from 6. The drug linker compound according to claim 5, wherein M is selected from 7. The drug linker compound according to any one of claims 1 to 4, wherein M is selected from 8. The drug linker compound according to any one of claims 1 to 4, wherein M is selected from 9. The drug linker compound according to any one of claims 1 to 8, wherein L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) r OCH3)), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, V al-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gl y-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys -Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Where R' represents hydrogen, C 1-6 an alkyl group or a polyethylene glycol segment containing 1 to 10 EO units; and s is an integer selected from 1 to 20.

10. The drug linker compound according to any one of claims 1 to 9, wherein L is selected from one or more of the following structures: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1-20.

11. The drug linker compound according to any one of claims 1 to 10, wherein L is selected from one or more of the following structures:

12. The drug linker compound according to any one of claims 1 to 11, wherein L is selected from the following structures:

13. The drug linker compound according to any one of claims 1 to 12, wherein L is selected from the following structures:

14. The drug linker compound according to any one of claims 1 to 13, wherein L is selected from the following structures:

15. The drug linker compound according to any one of claims 1 to 12, wherein L is selected from the following structures:

16. The drug linker compound according to any one of claims 1 to 15, wherein E is a single bond, -NHCH2-, The drug linker compound according to claim 16 , wherein E is —NHCH 2 —.

18. The drug linker compound according to any one of claims 1 to 17, Selected from the following structures:

19. The drug linker compound according to any one of claims 1 to 18, Selected from the following structures:

20. The drug linker compound according to any one of claims 1 to 19, wherein the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors.

21. The drug linker compound of any one of claims 1 to 20, wherein the tubulin inhibitor is an auristatin compound or a maytansine compound; the DNA intercalator is a pyrrolobenzodiazepine (PBD); the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); and the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

22. The drug linker compound according to any one of claims 1 to 21, wherein the cytotoxic drug is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of a compound of Formula I or Formula II: in, R1, R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ; where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

23. The drug linker compound of any one of claims 1 to 22, wherein the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

24. The drug linker compound of any one of claims 1 to 23, wherein the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

25. The drug linker compound according to any one of claims 1 to 24, wherein D is selected from the following structures:

26. The drug linker compound according to any one of claims 1 to 25, selected from the group consisting of A-01 to A-34, B-01 to B-07, and C-01 to C-28 shown below:

27. The drug linker compound of any one of claims 1 to 26, which is optionally substituted with one or more suitable substituents.

28. The drug linker compound of claim 27, having the following structure: in, R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, 5-12 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl, -C 1-6 Alkyl-C 6-10 Aryl and -C 1-6 Alkyl-5-12 membered heteroaryl; said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more selected from hydroxy, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1- 6 alkoxy, C 6-10 substituted by a substituent of an aryl group or a 5-12 membered heteroaryl group; R 13 and R 14 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and 4-6 membered heterocyclic group; the alkyl, cycloalkyl and heterocyclic group are optionally substituted by one or more selected from hydroxyl, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 6- 10 substituted by a substituent of an aryl group or a 5-12 membered heteroaryl group; R 15 Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, halogenated C 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 2-6 Alkenyl, C 2- 6-membered alkynyl and 3-6-membered heterocycloalkyl; R 16 H; or, R 15 、R 16 and the atoms to which they are connected together form a 4-7 membered ring; the 4-7 membered ring is optionally substituted by one or more selected from hydroxyl, CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 6-10 aryl, or a 5- to 12-membered heteroaryl substituent.

29. The drug linker compound of claim 28, having the following structure: R 10 、R 11 、R 12 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl and indolyl-C 1-6 alkyl-; R 13 and R 14 are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and 4-6 membered heterocyclic groups; R 15 Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, halogenated C 1-6 Alkyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 2-6 Alkenyl, C 2- 6-membered alkynyl and 3-6-membered heterocycloalkyl; R 16 H; or, R 15 、R 16 and the atoms to which they are connected together form a 4-7 membered ring. 。 30. Use of the drug linker compound according to any one of claims 1 to 29 in preparing a conjugate (eg, an antibody-drug conjugate).

31. A compound or a pharmaceutically acceptable salt thereof having the following structure: in, X is selected from benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, nitrobenzenesulfonyl, benzoyl, pivaloyl, trityl, 4-methoxyphenyldiphenylmethyl, dimethoxytrityl, 2,4-dimethoxybenzyl, p-methoxybenzyl and benzyl; a is an integer from 1 to 10, preferably an integer from 3 to 8; R1, R2 and D are as defined in any one of claims 1 or 20-25.

32. A compound having the following structure:

33. Use of the compound of claim 32 in the preparation of the drug linker compound of any one of claims 1 to 29.

34. A method for preparing a compound, comprising the step of deprotecting IM-5-a to obtain a compound of formula IM-6-a: in, X, R1 and R2 are as defined in claim 31 or 32.

35. The preparation method according to claim 34, wherein the deprotection reaction is carried out under one or more of the following conditions: (1) The solvent is selected from N,N-dimethylformamide; (2) adding an alkylamine compound to the reaction system; (3) Carry out at room temperature; (4) The reaction time is 1-5h.

36. The preparation method according to claim 34, further comprising the step of reacting IM-6-a with IM-2 to obtain compound A-14-a: in, R1 and R2 are as defined in claim 31 or 32.

37. The preparation method according to claim 36, wherein the reaction is carried out under one or both of the following conditions: (1) The reaction solvent is selected from N,N-dimethylformamide and N,N-dimethylacetamide; (2) The reaction further comprises the step of adding N,N-diisopropylethylamine.

38. A method for preparing a compound having the following structure: in, R1 and R2 are as defined in claim 31 or 32; a is an integer from 1 to 10; preferably, a is 3 or 8.

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