Camptothecin compounds and conjugates thereof

Abstract

Structural formula I: [Formula 1] The present invention provides a camptothecin compound represented by TIFF2024545233000241.tif51168 or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof.Antibody-drug conjugates comprising the camptothecin compound are also provided.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority from Chinese Patent Application No. 202111544686.7, filed on December 16, 2021, the entire contents of which are incorporated herein by reference for all purposes.

[0002] The present invention belongs to the field of biotechnology, and more particularly relates to camptothecin analogues having novel structures and their use in the preparation of pharmaceuticals, especially antibody-drug conjugates. [Background technology]

[0003] DNA topoisomerase is an essential enzyme that is widely present in living organisms, and is a general term for enzymes that catalyze the interconversion between DNA topoisomers. It is mainly divided into two types, namely, topoisomerase I (Topo I) and topoisomerase II (Topo II). Among them, topoisomerase I has high expression in various tumor cells, such as colon cancer cells, cervical cancer cells and ovarian cancer cells, and shows a content much higher than that in normal tissues or cells, and shows a significantly increased activity in tumor cells in the S phase. Therefore, an activity inhibitor that targets topoisomerase I can selectively inhibit DNA replication in tumor cells in the proliferation phase.

[0004] Studies have shown that topoisomerase I is the primary target of camptothecin (CPT) and its analogues. Camptothecin is a cytotoxic quinoline alkaloid that stabilizes the normally dissociated covalent complex between topoisomerase I and DNA strands to form a ternary complex. Once the ternary complex is formed, CPT exerts its anticancer effect by inhibiting DNA synthesis, thereby causing cell death by inhibiting the DNA cleavage / religation reaction initially mediated by topoisomerase I.

[0005] Due to the above properties, camptothecin and its analogs have become an important class of antitumor drugs, and are used as small molecule drugs in antibody-drug conjugates. Antibody-drug conjugates (ADCs) are novel therapeutic agents for tumor treatment, and generally consist of an antibody or antibody-like ligand, a small molecule drug, and a linker connecting the two. Antibody-drug conjugates (ADCs) combine the antitumor activity of small molecule drugs with the high selectivity and stability of antibodies or antibody-like ligands, as well as good pharmacokinetic properties, and are currently attracting attention in the field of tumor treatment.

[0006] To date, ADCs prepared using camptothecin (CPT) at home and abroad mainly include: 1. Sacituzumab govitecan (IMMU-132), an antibody-drug conjugate of DAR 8 targeting Trop-2, prepared by coupling the topoisomerase I inhibitor SN38 with the anti-Trop-2 antibody SAC via CL2A-SN38 (drug-containing linker). [ka] 2. Enhertu (DS-8201), developed by Daiichi Sankyo Co., Ltd., is an antibody-drug conjugate with DAR 8 prepared by coupling an exatecan analogue with the anti-HER2 antibody trastuzumab via MC-GGFG-Dxd (drug-containing linker). [ka] 3. DS-1062 and DS-7300 under investigation by Daiichi Sankyo Co., Ltd. DS-1062 is a DAR 4 antibody-drug conjugate prepared by coupling an exatecan analogue to the anti-Trop-2 antibody hTINA1 via MC-GGFG-Dxd (a drug-containing linker) using stochastic conjugation. DS-7300 is a DAR 4 antibody-drug conjugate targeting B7H3 prepared by coupling MC-GGFG-Dxd (a drug-containing linker) to the anti-B7H3 antibody hM30 using stochastic conjugation. [ka]

[0007] While the above-mentioned ADCs show some therapeutic effects, they have certain problems. For example, due to the chemical instability of the carbonate bond in IMMU-132, IMMU-132 shows a half-life in plasma of only about 12 hours, and causes problems such as increased side effects that cause patients to have diarrhea, fatigue, nausea, febrile neutropenia, leukopenia and other toxic reactions, although to a greater or lesser extent (Patent Document 1). For DS-1062 and DS-7300, the stochastic conjugation mode causes a fairly large heterogeneity of ADC products.

[0008] Currently, there are only a few types of camptothecin-containing ADCs available, and they have disadvantages such as a narrow range of target populations, low therapeutic efficacy of single agents, strong toxicity and side effects, etc. Therefore, there is still a need to develop new camptothecins and corresponding ADCs to meet therapeutic needs. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2014 / 0170063 Summary of the Invention

[0010] In view of the above problems, an object of the present disclosure is to provide a compound having a novel structure, which is a camptothecin compound itself or a compound formed by linking a camptothecin compound to a linker, and an antibody-drug conjugate produced using the same.

[0011] In the context of this disclosure, halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0012] In the context of the present disclosure, the terms "linker" and "linker compound" are used interchangeably.

[0013] In the context of the present disclosure, the term "drug-containing linker" refers to a compound obtained by directly or indirectly covalently attaching a drug (e.g., a small molecule drug such as a camptothecin compound) to a linker.

[0014] In the context of the present disclosure, when a group is substituted, it may be substituted by one or more substituents, the number of substituents being determined by the number of hydrogen atoms which the group contains, all of which can be substituted.

[0015] The present disclosure provides the following technical solutions:

[0016] In a first aspect, the disclosure provides camptothecin compounds.

[0017] In a first embodiment of the disclosure, the camptothecin compound has structural formula I: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0018] In structural formula I, unless otherwise specified in the context of this disclosure, R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3 and R4 together with the carbon atom to which they are attached form a C3-6 cycloalkyl. When R1, R2, R3 and R4 are each independently C1-6 alkoxy, the C1-6 alkoxy includes linear or branched C1-6 alkoxy, preferably linear or branched C1-3 alkoxy, more preferably methoxy. When R1, R2, R3 and R4 are each independently substituted amino, the substituted amino is an amino substituted with one or more substituents selected from the group consisting of methyl and ethyl. When R1, R2, R3 and R4 are each independently a C1-7 alkyl or a substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes a straight-chain or branched C1-7 alkyl or a substituted C1-7 alkyl, the substituted C1-7 alkyl being a C1-7 alkyl substituted with one or more substituents selected from the group consisting of cyclopropyl and cyclobutyl, or the straight-chain or branched C1-7 alkyl or substituted C1-7 alkyl is preferably a C1-3 alkyl or a substituted C1-3 alkyl, such as methyl and halomethyl (preferably trifluoromethyl).

[0019] In formula I, unless otherwise specified in the context of the present disclosure, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0020] In structural formula I, unless otherwise specified in the context of this disclosure, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. In the substituted methylene, either hydrogen of the methylene may be replaced or both hydrogens of the methylene may be replaced with a substituent which may be benzyl or alkyl, and when the substituent is alkyl, the alkyl and R3 and / or R4 may form a C3-6 fused ring structure or a spiro ring structure together with the carbon atom to which they are attached. When Y is a substituted methylene, the substituted methylene is preferably a methylene substituted with an alkyl, more preferably a straight chain or branched C1-4 alkyl. Preferably, Y is oxygen, sulfur, sulfone or sulfoxide, or preferably, Y is oxygen, sulfur or methylene.

[0021] In structural formula I, X is oxygen or sulfur, unless otherwise indicated in the context of this disclosure.

[0022] In structural formula I, n=0 or 1, unless otherwise specified in the context of this disclosure.

[0023] In structural formula I, when R1, R2, R3 and R4 are simultaneously hydrogen, X is oxygen, and n=0, then when Y is methylene, G cannot be hydrogen or fluorine, and when Y is oxygen or sulfur, G cannot be hydrogen.

[0024] Preferably, R1, R2, R3 and R4 are each independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3 and R4 together with the carbon atom to which they are attached form a C3-6 cycloalkyl (e.g., C3-5 cycloalkyl). Furthermore, R1 and R2 may be the same, and / or R3 and R4 may be the same.

[0025] Preferably, Y is methylene substituted with alkyl, and the alkyl and R3 and / or R4 may form a C3-6 fused ring structure or a spiro ring structure together with the carbon atom to which they are attached.

[0026] Preferably, X may be oxygen.

[0027] Preferably, X is oxygen, G is hydrogen, halogen (eg fluorine or chlorine), methyl or methoxy, and Y and R1, R2, R3 and R4 are defined as above.

[0028] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3 and R4 are defined as above.

[0029] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3 and R4 are defined as above.

[0030] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3 and R4 are defined as above.

[0031] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3 and R4 are defined as above.

[0032] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3 and R4 are defined as above.

[0033] Preferably, X is oxygen, G is hydrogen, Y is oxygen, sulfone or sulfoxide, and R1, R2, R3 and R4 are defined as above.

[0034] Preferably, X is oxygen, G is hydrogen, Y is sulfone or sulfoxide, and R1, R2, R3 and R4 are defined as above.

[0035] Compounds represented by formula I provided by the present disclosure may further be represented by formula IA: [ka] Preferably, the compound is represented by:

[0036] In formula IA, the groups R1, R2, R3 and R4 have the same meanings as defined above for the groups R1, R2, R3 and R4 in formula I, except that R1, R2, R3 and R4 are not simultaneously hydrogen.

[0037] Alternatively, in the first aspect of the disclosure, the camptothecin compound has the structural formula II: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0038] In structural formula II, unless otherwise specified in the context of this disclosure, R5 is C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cycloalkyl or C3-6 cycloalkyl substituted with one or more substituents, phenyl or substituted phenyl. When R5 is C1-5 alkyl or substituted C1-5 alkyl, C1-5 alkyl includes linear or branched C1-5 alkyl. Furthermore, R5 is C1-4 linear alkyl. When R5 is substituted C1-5 alkyl or substituted C3-6 cycloalkyl, substituted C1-5 alkyl or substituted C3-6 cycloalkyl is C1-5 alkyl or C3-6 cycloalkyl substituted with substituent(s) selected from the group consisting of halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methanesulfonyl and C3-6 cycloalkyl, and among the substituent(s), substituted amino is amino substituted with one or more substituents selected from the group consisting of methyl and ethyl. When R5 is a substituted phenyl, the substituted phenyl is phenyl substituted with a substituent(s) selected from the group consisting of alkyl (eg, C1-6 alkyl, preferably C1-3 alkyl) and halogen.

[0039] In formula II, unless otherwise specified in the context of the present disclosure, G is hydrogen, halogen (e.g., fluorine), methyl or methoxy. G is preferably hydrogen, fluorine or chlorine.

[0040] In structure II, X is oxygen or sulfur.

[0041] In structure II, n=0 or 1.

[0042] In structure II, when X is oxygen, G is hydrogen, and n=0, R5 cannot be n-butyl.

[0043] Compounds represented by formula II provided by the present disclosure may further be represented by formula IIA: [ka] Preferably, the compound is represented by:

[0044] In formula IIA, the group R5 has the same meaning as defined above for the group R5 in formula II, except that R5 cannot be n-butyl.

[0045] According to certain embodiments of the present disclosure, in a first aspect of the present disclosure, the compound has the following structure: [ka] [ka] [ka] [ka] It has one of the following.

[0046] In a second aspect, the disclosure provides a drug-containing linker having a structure represented by the general formula "LA-CPT", where L represents a linker used in an antibody-drug conjugate (ADC), A represents a peptide group of one or more amino acids, and CPT is a compound of camptothecin.

[0047] In a second embodiment of the present disclosure, a drug-containing linker having a structure represented by the general formula "LA-CPT" is represented by structural formula III: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0048] In structural formula III, unless otherwise indicated in the context of this disclosure, E is [ka] means the bonding site with M, [ka] is selected from the group consisting of:

[0049] In structural formula III, unless otherwise specified in the context of the present disclosure, M is phenylene or phenylene substituted with one or more substituents, or a chemical bond, where in the substituted phenylene, the phenylene is substituted with a substituent(s) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, e.g., trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano, and preferably, M is halogen-substituted phenylene.

[0050] In structural formula III, unless otherwise specified in the context of the present disclosure, SP1 is selected from the group consisting of C1-8 alkylene, C1-8 cycloalkylene, or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene containing 1-11 (preferably 1-6) heteroatoms selected from the group consisting of N, O, and S, wherein C1-8 alkylene, C1-8 cycloalkylene, and C1-21 straight chain heteroalkylene are each independently optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano.

[0051] In formula III, unless otherwise specified in the context of this disclosure, SP2 is -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) aor a chemical bond, where a is an integer in the range of 1-20, preferably an integer in the range of 1-10, and more preferably an integer in the range of 1-6.

[0052] In structural formula III, unless otherwise specified in the context of the present disclosure, A means a peptide group of 2 to 4 amino acids. When A means a peptide group of 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO. When A means a peptide group of 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO. When A denotes a peptide group of four amino acids, this may be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO.

[0053] In structural formula III, CPT is a compound of camptothecin, unless otherwise specified in the context of this disclosure.

[0054] When group E is E1, formula III provided by the present disclosure can further be represented by formula IIIA: [ka] It is.

[0055] In structural formula IIIA, unless otherwise specified in the context of this disclosure, R6 and R7 are each independently hydrogen, halogen, or Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents, where the phenyl is substituted with a substituent(s) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano. Ar' is phenyl, phenyl substituted with 4-formylmethylamine ( [ka] ) or phenyl substituted with 4-formylmorpholine ( [ka] ) is preferred.

[0056] For example, in formula III and formula IIIA, CPT is a compound represented by formula I or formula IA, or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and one of the corresponding specific compounds, all of which are provided in the first aspect of the present disclosure above.

[0057] CPT is represented by structural formula I: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in structural formula I, the groups G, X, Y, R1, R2, R3, R4 and n have the same meanings as defined above in the first aspect for the groups G, X, Y, R1, R2, R3, R4 and n in structural formula I.

[0058] CPT has structural formula IA as provided in the first aspect of the present disclosure above: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in structural formula IA, the groups R1, R2, R3 and R4 have the same meanings as defined above in the first aspect for the groups R1, R2, R3 and R4 in structural formula IA, except that R1, R2, R3 and R4 may simultaneously be hydrogen.

[0059] In particular, when "CPT" in the compound represented by structural formula IIIA is a compound represented by structural formula IA or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 may or may not be hydrogen at the same time. Similarly, R1, R2, R3 and R4 in structural formula IA may or may not be hydrogen at the same time. According to certain embodiments of the present disclosure, when R6 and R7 are hydrogen at the same time, R1, R2, R3 and R4 in structural formula IA cannot be hydrogen at the same time, and when R6 and R7 are not hydrogen at the same time, R1, R2, R3 and R4 in structural formula IA may or may not be hydrogen at the same time.

[0060] In formula III and formula IIIA, the compound represented by formula I or formula IA is bonded to the carboxyl of A by an amide bond through its amino (shown in formula I or formula IA, respectively), i.e., an amide bond is formed between the amino of the compound represented by formula I or formula IA and the carboxyl of A in formula III or formula IIIA.

[0061] For another example, in formula III and formula IIIA, CPT is a compound represented by formula II or formula IIA, or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and one of the corresponding specific compounds, all of which are provided in the first aspect of the present disclosure above.

[0062] CPT is represented by structural formula II as provided in the first aspect of the present disclosure above: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in structural formula II, the groups G, R5, X and n have the same meanings as defined above in the first aspect for the groups G, R5, X and n in structural formula II.

[0063] CPT has structural formula IIA as provided in the first aspect of the present disclosure above: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in structural formula IIA, the group R5 has the same meaning as defined above in the first aspect for the group R5 in structural formula IIA, except that the group R5 can be n-butyl.

[0064] In formula III and formula IIIA, the compound represented by formula II or formula IIA is bonded to the carboxyl of A by an amide bond via its amino (shown in formula II or formula IIA, respectively), i.e., an amide bond is formed between the amino of the compound represented by formula II or formula IIA and the carboxyl of A in formula III or formula IIIA.

[0065] Each of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 14-P, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, Compound 38, Compound 39 and Compound 40 provided above, or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, can be used as "CPT" in Structural Formula III, and is bonded to the carboxyl of A in Structural Formula III or Structural Formula IIIA by an amide bond via its amino.

[0066] As another example, in formula III and formula IIIA, CPT can be represented by formula IV: [ka] or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0067] In structural formula IV, unless otherwise indicated in the context of this disclosure, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cycloalkyl or C3-6 cycloalkyl substituted with one or more substituents, or halogen.

[0068] When R8 is a substituted C1-5 alkyl or a substituted C3-6 cycloalkyl, the C1-5 alkyl or C3-6 cycloalkyl is substituted with a substituent (which may be a plurality of substituents) selected from the group consisting of halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methanesulfonyl, and C3-6 cycloalkyl, and among the substituents, the substituted amino is amino substituted with one or more substituents selected from the group consisting of methyl and ethyl.

[0069] In particular, when "CPT" in the compound represented by structural formula IIIA is a compound represented by structural formula IV or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, R6 and R7 may or may not be hydrogen at the same time. Similarly, R8 in structural formula IV may or may not be hydrogen. According to certain embodiments of the present disclosure, when R6 and R7 are hydrogen at the same time, R8 may or may not be hydrogen.

[0070] In the formula III and the formula IIIA, the compound represented by the formula IV is bonded to the carboxyl of A by a self-releasing structure through its hydroxyl bonded to the same carbon as R8 (shown in the formula IV), and the self-releasing structure is, for example, ( [ka] ) where the solid line indicates the site of attachment to the carboxyl group of A in structural formula III or structural formula IIIA; [ka] denotes the bonding site with the hydroxyl in structure IV.

[0071] In particular, with respect to compounds represented by Structural Formula IIIA, when R6 and R7 are Ar'S, M is preferably phenylene or substituted phenylene, while SP1 is a C1-21 (preferably C1-16, more preferably C1-11) linear heteroalkylene containing 1 to 11 (preferably 1 to 6) heteroatoms selected from the group consisting of N, O, and S.

[0072] Preferably, the compounds represented by formula III or formula IIIA provided by the present disclosure further comprise formula V: [ka] It is a compound represented by:

[0073] In formula V, R6 and R7 are each independently Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents, where the phenyl is substituted with a substituent(s) selected from the group consisting of alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano. Ar' is phenyl, phenyl substituted with 4-formylmethylamine ( [ka] ) or phenyl substituted with 4-formylmorpholine ( [ka] ) is preferred.

[0074] In structural formula V, unless otherwise specified in the context of the present disclosure, Xh and Yh are each independently hydrogen, halogen, haloalkyl (e.g., C1-C6 haloalkyl, preferably C1-C4 haloalkyl, such as trifluoromethyl), or alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

[0075] In Structural Formula V, m is any integer ranging from 1 to 10, preferably 1 to 5, and more preferably 3 to 5, unless otherwise specified in the context of this disclosure.

[0076] In formula V, A represents a peptide group of 2 to 4 amino acids as defined above.

[0077] In formula V, the meaning of CPT and its bond relationship in formula V are the same as those of CPT in formula III and formula IIIA defined above.

[0078] Preferably, the compound represented by formula V provided by the present disclosure further comprises the compound represented by formula VA: [ka] It is a compound represented by:

[0079] In formula VA, the groups A, G, Y, R1, R2, R3, R4, X and n have the same meanings as defined above for the groups A, G, Y, R1, R2, R3, R4, X and n in formula III or formula IIIA.

[0080] In formula VA, G is preferably hydrogen, fluorine or chlorine.

[0081] In formula VA, Y is preferably methylene, sulfur or oxygen.

[0082] In structural formula VA, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0083] Preferably, the compound represented by structural formula VA provided by the present disclosure further comprises structural formula VA-1: [ka] It is a compound represented by:

[0084] Alternatively, the compounds represented by formula V provided by the present disclosure may further comprise formula VB: [ka] It is a compound represented by:

[0085] In formula VB, the groups A, G, R5, X and n have the same meanings as defined above for the groups A, G, R5, X and n in formula III or formula IIIA.

[0086] In structural formula VB, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0087] Preferably, the compound represented by formula VB provided by the present disclosure further comprises formula VB-1: [ka] It is a compound represented by:

[0088] Alternatively, the compounds represented by formula V provided by the present disclosure may further comprise the compound represented by formula VC: [ka] It is a compound represented by:

[0089] In formula VC, the groups A and R8 have the same meanings as defined above for the groups A and R8 in formula III or formula IIIV.

[0090] In structural formula VC, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0091] In structural formula VC, unless otherwise indicated in the context of this disclosure, " [ka] " is the same as the self-release structure described above when the CPT in structural formula III or structural formula IIIV is represented by structural formula IV.

[0092] According to certain embodiments of the present disclosure, the compound represented by structural formula V provided by the present disclosure may further comprise structural formula VI: [ka] It is a compound represented by:

[0093] In formula VI, A represents a peptide group of 2 to 4 amino acids as defined above.

[0094] In formula VI, CPT is a compound of camptothecin. The meaning of CPT and its bond relationship in formula VI are the same as those of CPT in formula III and formula IIIA defined above.

[0095] Preferably, the compound represented by formula VI provided by the present disclosure further comprises formula VI-A: [ka] It is a compound represented by:

[0096] In structural formula VI-A, the groups A, G, Y, R1, R2, R3, R4, X, and n have the same meanings as defined above for the groups A, G, Y, R1, R2, R3, R4, X, and n in structural formula III or structural formula IIIA.

[0097] In formula VI-A, G is preferably hydrogen, fluorine or chlorine.

[0098] In formula VI-A, Y is preferably methylene, sulfur or oxygen.

[0099] In structural formula VI-A, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0100] Preferably, the compound represented by formula VI-A provided by the present disclosure further comprises the compound represented by formula VI-A-1: [ka] It is a compound represented by:

[0101] Alternatively, the compound represented by formula VI may further be represented by formula VI-B: [ka] It is a compound represented by:

[0102] In formula VI-B, the groups A, G, R5, X and n have the same meanings as defined above for the groups A, G, R5, X and n in formula III or formula IIIA.

[0103] In structural formula VI-B, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino of CPT and the carboxyl of A.

[0104] More preferably, the compound represented by formula VI-B further comprises the compound represented by formula VI-B-1: [ka] It is a compound represented by:

[0105] Alternatively, the compounds represented by formula VI provided by the present disclosure may further comprise formula VI-C: [ka] It is a compound represented by:

[0106] In formula VI-C, the groups A and R8 have the same meanings as defined above for the groups A and R8 in formula III or formula IIIA.

[0107] In structural formula VI-C, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0108] The self-emitting structure in the structural formula VC is [ka] If, then structure VC may further be structure VI-C.

[0109] According to certain embodiments of the present disclosure, in a second aspect of the present disclosure, the compound has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It has one of the following.

[0110] In a third aspect, the present disclosure provides an antibody-drug conjugate prepared using a compound represented by structural formula I, structural formula IA, structural formula II, structural formula II-A, structural formula III, structural formula III-A, structural formula V, structural formula VA, structural formula VA-1, structural formula VB, structural formula VB-1, structural formula VC, structural formula VI, structural formula VI-A, structural formula VI-A-1, structural formula VI-B, structural formula VI-B-1 or structural formula VI-C, or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and an antibody or a fragment thereof.

[0111] In a third embodiment of the present disclosure, the antibody or fragment thereof is directed to a tumor associated antigen, such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c- Targets MET, FGFR, nectin 4, AGS-16, guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3 or claudin 18.2.

[0112] Preferably, the antibody-drug conjugate has the general formula: [ka] (wherein mAb means an antibody or a fragment thereof; The groups M, SP1, SP2, A and CPT have the same meaning as defined above for the groups M, SP1, SP2, A and CPT in formula III or formula IIIA. N ranges from 1 to 10, preferably 1 to 8 (e.g., 1 to 5), and more preferably 3 to 8.

[0113] In the above general formula, E L teeth, [ka] means binding to cysteine ​​in the mAb, [ka] represents a bond to M, [ka] is selected from the group consisting of:

[0114] In the above general formula, the mAb may be an antibody or fragment thereof of the IgG type, preferably an antibody or fragment thereof of the IgG1 subtype, that targets any of the above tumor-associated antigens.

[0115] Preferably, when the compound represented by structural formula VI is conjugated to an antibody or fragment thereof, the antibody-drug conjugate has the general formula VII or general formula VIII as follows: [ka] The compound has a structure represented by:

[0116] In the general formulae VII and VIII, N is 1-10, preferably 1-8 (eg, 1-5), and more preferably 3-8.

[0117] In the general formula VII and the general formula VIII, Ab is represented by the above general formula: [ka] This corresponds to the mAb in

[0118] In general formula VII and general formula VIII, the groups A and CPT have the same meanings as defined above in the second embodiment for the groups A and CPT in formula III or formula IIIA.

[0119] In a third aspect, the antibody-drug conjugates represented by general formula VII or VIII provided by the present disclosure can produce prodrug metabolites in cells, such as tumor cells.

[0120] In a fourth aspect, the present disclosure provides a compound of structural formula IX: [ka] The present invention provides a compound represented by the formula:

[0121] In formula IX, the groups A and CPT have the same meanings as defined above in the second embodiment for the groups A and CPT in formula III or formula IIIA.

[0122] Specifically, the prodrug metabolite of the antibody-drug conjugate made using the compound represented by structural formula VI-A has the structure represented by structural formula IX-A: [ka] It is a compound represented by:

[0123] In structural formula IX-A, the groups A, G, Y, R1, R2, R3, R4, X and n have the same meanings as defined above in the second aspect for the groups A, G, Y, R1, R2, R3, R4, X and n in structural formula III or structural formula IIIA.

[0124] In structural formula IX-A, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0125] Preferably, the compound represented by structural formula IX-A provided by the present disclosure further comprises structural formula IX-A-1: [ka] It is a compound represented by:

[0126] The prodrug metabolites of the antibody-drug conjugates made using the compounds represented by structural formula VI-B are represented by structural formula IX-B: [ka] It is a compound represented by:

[0127] In formula IX-B, the groups A, G, R5, X and n have the same meanings as defined above in the second embodiment for the groups A, G, R5, X and n in formula III or formula IIIA.

[0128] In structural formula IX-B, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0129] The prodrug metabolite of the antibody-drug conjugate made using the compound represented by structural formula VI-C has the structure represented by structural formula IX-C: [ka] It is a compound represented by:

[0130] In formula IX-C, the groups A and R8 have the same meanings as defined above in the second embodiment for the groups A and R8 in formula III or formula IIIA.

[0131] In structural formula IX-C, unless otherwise specified in the context of this disclosure, "-A-NH-" means that an amide bond is formed between the amino and the carboxyl of A.

[0132] In a fifth aspect, the present disclosure provides the use of a compound according to the invention or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, or an antibody-drug conjugate in the manufacture of a medicament for the treatment of a tumor.

[0133] Preferably, the tumor is a cancer. Preferably, the tumor expresses a tumor associated antigen, such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue Factor, c-MET, FGFR, Nectin 4, AGS-16 , guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3 or claudin 18.2, or are associated with high expression of tumor-associated antigens.

[0134] The tumor is preferably colon cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, or glioblastoma.

[0135] In a sixth aspect, the present disclosure provides a method of treating a tumor with a compound or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, or an antibody-drug conjugate according to the present invention, comprising administering to a subject in need thereof the compound or a pharma- ceutically acceptable salt, stereoisomer, solvate or prodrug thereof, or the antibody-drug conjugate.

[0136] Preferably, the tumor is a cancer. Preferably, the tumor expresses a tumor associated antigen, such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue Factor, c-MET, FGFR, Nectin 4, AGS-16 , guanylyl cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, delta-like protein 3 or claudin 18.2, or are associated with high expression of tumor-associated antigens.

[0137] The tumor is preferably colon cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, or glioblastoma.

[0138] Preferably, the subject is a mammal, preferably a primate, more preferably a human.

[0139] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will now be described in detail with reference to the accompanying drawings. [Brief description of the drawings]

[0140] [Figure 1] FIG. 1 shows the results of analysis of an antibody-drug conjugate according to the present invention by hydrophobic interaction chromatography (HIC). [Diagram 2] FIG. 1 shows the results of a study of the efficacy of an antibody-drug conjugate according to the present invention in a mouse model of pancreatic cancer. [Diagram 3] FIG. 1 shows the results of a study of the efficacy of an antibody-drug conjugate according to the present invention in a mouse model of bladder cancer. [Figure 4] FIG. 1 shows the results of a study of the efficacy of an antibody-drug conjugate according to the present invention in a mouse model of lung cancer. [Diagram 5] FIG. 1 shows the results of a bystander effect study of antibody-drug conjugates according to the present invention (mean±SD, n=2). [Figure 6] FIG. 1 shows time-effect curves of internalization of antibody-drug conjugates and antibodies according to the present invention (mean±SD, n=3). [Figure 7] FIG. 1 shows the results of a study on the induction of apoptosis in tumor cells by antibody-drug conjugates according to the invention. [Figure 8] FIG. 1 shows dose-response curves of binding of an antibody-drug conjugate and an antibody according to the present invention to tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] The present invention will now be described with reference to specific examples. It will be appreciated by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.

[0142] All experimental procedures in the following examples are conventional unless otherwise indicated. All materials and reagents used in the following examples are commercially available unless otherwise indicated. EXAMPLES

[0143] Group of Examples 1 Synthesis of Camptothecin Common methods for synthesizing camptothecin: The camptothecin compounds according to the present invention can be obtained by Friedlander reaction using a compound represented by formula A and a tricyclic compound (cyclic compound CDE), the general reaction being as shown below: [ka]

[0144] In the reaction, the tricyclic compound CDE: [ka] can be purchased from MCE (MedChemExpress).

[0145] Tricyclic compound HCDE: [ka] can be obtained according to the method described in Bioorganic & Medicinal Chemistry, 2010, vol. 18, # 9, p. 3140-3146.

[0146] Example Group 1.1 Synthesis of Compounds Represented by Formula A Example 1.1.1 Synthesis of Compound A1 [ka]

[0147] Method 1: Compound A1 was synthesized according to the method described in WO2020200880. The synthetic route is shown below: [ka]

[0148] Method 2: Compound A1 was synthesized by palladium-catalyzed coupling reaction using zinc reagent. The synthetic route is shown below: [ka]

[0149] (1) Acetylation: 3-Bromo-4-nitroaniline (25.00 g, 0.12 mol) and 200 mL of acetic acid were added to a 500 mL flask, followed by slow dropwise addition of 50 mL of acetic anhydride. These starting compounds were reacted at room temperature for 16 hours. After completion of the reaction as confirmed by TLC detection, the reaction solution was filtered to obtain a filtrate, which was then concentrated under reduced pressure to remove acetic acid. The resulting filter cake was pooled and slurried with 200 mL of MTBE, and the slurry was filtered to obtain 27.6 g of a dried yellow solid (Intermediate A1-a) in 92% yield. LC-MS (ESI): m / z 259 (M+H)+.

[0150] (2) Preparation of 4-ethoxy-4-oxobutylzinc bromide Activated zinc powder (19.0 g, 0.29 mol, 2.00 equiv.) was added to a 250 mL three-neck flask (equipped with a thermometer, reflux condenser, and rubber stopper). The air in the flask was then replaced with nitrogen, and anhydrous DMF (145 mL) was added, after which the air in the flask was replaced with nitrogen again. Iodine (1.86 g, 0.015 mol, 0.1 equiv.) was added at room temperature, and the color of the solution was observed to change from colorless to brownish red, gradually to pale yellow, and finally to colorless (2 to 3 min.). Next, ethyl 4-bromobutyrate (28.6 g, 0.15 mol, and 1.00 equiv.) was added to the flask, which was then heated to 80° C. (internal temperature) and reacted for 4 to 5 hours. When the reaction was complete as confirmed by TLC detection, the reaction solution was allowed to stand and cooled to room temperature for later use. The resulting supernatant was pale yellow in color and had a concentration of 1 mol / L.

[0151] (3) Coupling with zinc reagents Anhydrous DMF (120 mL) and intermediate A1-a (25.0 g, 1.00 eq.) were added to a 500 mL reaction flask. The air in the flask was then replaced with nitrogen, palladium acetate (433 mg, 0.02 eq.) was added, and the air in the flask was replaced with nitrogen again. The mixture in the flask was stirred at room temperature for 10 minutes, after which S-PHOS (1.6 g, 0.04 eq.) was added, and then the air in the flask was replaced with nitrogen again. Next, the mixture in the flask was stirred for 20 minutes, and 4-ethoxy-4-oxobutylzinc bromide (145 mL, 1.50 eq.) prepared in the above step was added dropwise at room temperature (25°C to 30°C), and the reaction was maintained at 25°C to 30°C for 16 hours. When the starting compound was confirmed to have reacted completely by TLC detection (DCM:EA=5:1), the reaction solution was cooled to room temperature and ammonium chloride solution (15 mL) was added to the reaction solution to quench the reaction. The reaction solution was then poured into 1 L of water, and 400 mL of ethyl acetate was added to obtain separate phases. The phases were filtered through a Buchner funnel, and the resulting aqueous phase was then extracted with ethyl acetate (400 mL×2), after which the resulting organic phase was washed twice with water (500 mL) and once with saturated sodium chloride solution (500 mL), followed by drying over anhydrous sodium sulfate and concentrating under reduced pressure to obtain 37.0 g of a reddish-brown oily liquid (Intermediate A1-b) in 130% yield. The crude product was used in the next reaction without further purification. LC-MS (ESI): [M+1]+=295.

[0152] (4) Synthesis of intermediate A1-c Intermediate A1-b (37.0 g, 1.0 equiv.) and ethanol (1.5 L) were added to a 3 L three-necked reaction flask, and the starting material was completely dissolved in ethanol. The reaction was cooled to 5° C., 5% Pd / C (5.7 g, 1.0 equiv.) was added, and the temperature was raised to 25° C. under hydrogen atmosphere (atmospheric pressure) and reacted for 16 h. When the reaction was complete by TLC detection (DCM:EA=5:1), the reaction was filtered through Celite, and the resulting organic phase was concentrated to give 33.0 g of crude product (Intermediate A1-c) in 130% yield. The crude product was used directly in the next reaction without further purification. LC-MS (ESI): [M+1]+=265.

[0153] (5) Synthesis of intermediate A1-d Intermediate A1-c (33.0 g, 1.00 equiv.) and 260 mL of acetic acid were added to a 1 L flask, and then 46 mL of acetic anhydride was slowly added dropwise thereto. The starting material compound was reacted at room temperature for 2 hours. When the starting compound was confirmed to have reacted completely by TLC detection (DCM:MeOH=10:1), the reaction solution was filtered to obtain a filtrate, which was then concentrated under reduced pressure to remove acetic acid. 250 mL of water was added, followed by extraction of the resulting aqueous phase with ethyl acetate (400 mL×2), after which the resulting organic phase was washed twice with water (500 mL) and once with saturated sodium chloride solution (500 mL), followed by drying with anhydrous sodium sulfate and concentration under reduced pressure to obtain 32 g of crude product. The crude product was slurried in 400 mL of MTBE, filtered, and 27.0 g of dried pale yellow solid (Intermediate A1-d) was obtained in 91% yield. The overall yield for the four steps was found to be 83.7%. LC-MS(ESI):[M+1]+=307. 1H NMR(400MHz,DMSO) δ 9.87(s,1H), 9.19(s,1H), 7.41(s,1H), 7.37(d,J=8.7Hz,1H), 7.20(d,J=8.5Hz,1H), 4.06(q,J=7.0Hz,2 H), 2.52(d,J=8.5Hz,2H), 2.29(t,J=7.3Hz,2H), 2.02(s,6H), 1.79~1.64(m,2H), 1.18(t,J=7.1Hz,3H).

[0154] (6) Synthesis of intermediate A1-e Intermediate A1-d (27.0 g, 88 mmol, 1.0 equiv), water (100 mL) and tetrahydrofuran (200 mL) were added to a 500 mL three-necked reaction flask to completely dissolve the starting materials. Lithium hydroxide monohydrate (18.5 g, 441 mmol, 5.0 equiv) was added at room temperature and the reaction was maintained at room temperature for 3 h. When the reaction was complete as detected by TLC, most of the tetrahydrofuran was distilled off under reduced pressure and 300 mL of water was added to the resulting residue. The resulting aqueous phase was extracted with EA (2×100 mL), the resulting organic phase was discarded, and the resulting aqueous phases were pooled, placed in an ice bath and adjusted to pH 4 by adding 6N hydrochloric acid. A solid precipitated and was filtered to give 19.1 g of a white solid (Intermediate A1-e) in 78% yield. 1H NMR(400MHz,DMSO) δ 12.09(s,1H), 9.86(s,1H), 9.18(s,1H), 7.38(d,J=15.5Hz,2H), 7.23(d,J=8.5Hz ,1H), 2.51(d,J=8.1Hz,2H), 2.23(t,J=7.1Hz,2H), 2.02(s,6H), 1.76~1.61(m,2H)

[0155] (7) Synthesis of Compound A1 40 mL of polyphosphoric acid was added to a 250 mL reaction flask and heated to 90 ° C, and then intermediate A1-e (5.0 g, 17.97 mmol) was added in several portions. The internal temperature was maintained at 95 ° C to 100 ° C and the reaction was allowed to proceed for 5 hours. When the completion of the reaction was confirmed by TLC detection, the heating source was removed and the reaction system was cooled to 50 ° C to 60 ° C. Then, 15 mL of 4 M HCl (aqueous solution) was added dropwise to the reaction system to quench the reaction (the temperature rose to 100 ° C). Then, 600 mL of 4 M NaOH aqueous solution was added dropwise to adjust the pH to 10. After the aqueous phase was extracted with ethyl acetate (50 mL x 3), the organic phases were pooled and washed once with saturated sodium chloride solution (50 mL), then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 3.75 g of a yellow solid (Intermediate A1-f) in 80.5% yield. The solid was used directly in the next reaction without purification. LC-MS (ESI): [M+1]+ = 261.

[0156] Starting material intermediate A1-f (3.75 g) was suspended in 19% hydrochloric acid (30 mL) in a 250 mL three-necked reaction flask. The reaction was heated to 90° C. (internal temperature) and reacted for 3 h. When the reaction was complete by TLC detection, the flask was placed in an ice-salt bath and cooled to below 5° C. 4 M NaOH solution (45 mL, 30 equiv.) was added dropwise to adjust the pH to 10. After the obtained aqueous phase was extracted with ethyl acetate (80 mL×5), the obtained organic phases were pooled and washed once with saturated sodium chloride solution (50 mL), followed by drying over anhydrous sodium sulfate and concentration under reduced pressure to obtain 2.45 g of crude product. After purifying the crude product by column chromatography using DCM as eluent, 1.93 g of yellow solid (compound A1) was obtained in 76% yield. The overall yield for the two steps was found to be 61.2%. LC-MS(ESI):[M+1]+=177. 1H NMR(400MHz,DMSO) δ 6.76(d,J=8.7Hz,1H), 6.68(s,2H), 6.42(d,J=8.7Hz,1H), 4.17(s,2H), 2.55(t,J=5.9Hz,2H), 2.46(t,J=6.2Hz,2H), 2.00~1.80(m,2H)

[0157] Method 3: Compound A1 was synthesized by lactam hydrolysis. The synthetic route is shown below: [ka]

[0158] The aminolactam compound was prepared according to the method described in Chinese Patent Publication No. 106349233. [ka]

[0159] The aminolactam compound (17.6 g, 0.1 mmol), ethanol (250 mL) and 98% sulfuric acid (5 mL) were mixed in a 500 mL three-neck flask, heated under reflux and reacted for 24 hours. The reaction solution was sampled and detected to confirm whether the reaction was complete. When the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure. Dichloromethane (200 mL) and water (100 mL) were added to the resulting residue, and the resulting mixture was placed in an ice bath and cooled to less than 10°C. After the pH was adjusted to 7-8 by adding 1N aqueous sodium hydroxide solution, the mixture was stirred to obtain separate phases. After the resulting aqueous phase was extracted with dichloromethane, the resulting organic phases were pooled and washed with saturated saline, followed by drying with anhydrous sodium sulfate, suction, and distillation under reduced pressure to remove the organic solvent, obtaining 25 g of crude product of diaminoethyl ester intermediate, which was used directly in the next reaction.

[0160] The diaminoethyl ester intermediate was dissolved in dichloromethane (200 mL) and triethylamine (20.2 g, 0.2 mol, 2 eq.) was added thereto. The mixture was placed in an ice bath and cooled to below 10° C., and acetic anhydride (25.5 g, 0.25 mol, 2.5 eq.) was added dropwise. The temperature was then maintained and reacted for 1 hour. The reaction solution was sampled and detected to see if the reaction was complete. Upon completion of the reaction, the reaction solution was poured into 1N ice-cold hydrochloric acid and stirred for 15 minutes. Separated phases were obtained, and the resulting aqueous phase was extracted with dichloromethane (50 mL×3), after which the resulting organic phase was pooled, dried over anhydrous sodium sulfate, sucked, and distilled under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was slurried in 400 mL of MTBE, then filtered and dried to obtain 26.9 g of intermediate A1-e as a pale yellow solid. The overall yield of the two steps was found to be 87.8%. LC-MS(ESI):m / z307(M+H) + .

[0161] Compound A1 was then prepared from intermediate A1-e according to Method 2.

[0162] Example 1.1.2 Synthesis of Compound A2 [ka] Compound A2 was synthesized according to the method described in WO2021148501. The synthetic route is shown below: [ka]

[0163] Example 1.1.3 Synthesis of Compound A3 [ka] Compound A3 was synthesized according to the method described in US Patent Publication No. 2004266803.

[0164] Example 1.1.4 Synthesis of Compound A4 Compound A4 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is shown below: [ka]

[0165] Step 1: Intermediate A4-2 was prepared from compound A4-1 following literature procedures.

[0166] Step 2: Intermediate A4-2 (3.4 g) was dissolved in dichloromethane at -5°C to 5°C, triethylamine (3.0 g) was added thereto, and then AllocCl (2.8 g) was slowly added dropwise. When the addition was completed, the resulting mixture was stirred for 1 to 2 hours to react, and then the reaction was quenched by adding water. The resulting reaction product was washed once with water and once with saturated saline, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 5.5 g of crude product of intermediate A4-3. LC-MS (ESI): [M+1]+=255.7.

[0167] Step 3: Intermediate A4-3 (5.5 g) was added to TBAF (55 ml) and acrylic acid (110 ml) at room temperature, and the resulting mixture was heated to 50°C to 55°C and stirred for 24 hours to react. The reaction solution was concentrated to dryness as it was, and purified by column chromatography using a mixture of petroleum ether to methanol / dichloromethane (1:50) as the eluent, to obtain about 5.2 g of crude product of intermediate A4-4. LC-MS (ESI): [M+1]+ = 327.4.

[0168] Step 4: Intermediate A4-4 (5.0 g) was added to 100 ml of a mixed solution of ethanol / water (3:1) at room temperature, and ammonium chloride (5.5 g) and iron powder (5.5 g) were added thereto. The resulting mixture was heated under reflux and reacted for 1 to 2 hours. The reaction solution was cooled to room temperature, filtered, and the resulting solid was washed with ethanol and concentrated to dryness to obtain about 3.8 g of a crude product of intermediate A4-5. LC-MS (ESI): [M+1]+ = 297.4.

[0169] Step 5: Intermediate A4-5 (3.8 g) was added to trifluoroacetic acid (38 ml) at 20°C to 30°C, and trifluoroacetic anhydride (38 ml) was added thereto. The resulting mixture was stirred and reacted for 18 to 24 hours, and then the resulting reaction product was subjected to the following work-up: it was directly concentrated to dryness and purified by column chromatography using a mixture of petroleum ether / ethyl acetate (1:0 to 4:1) as the eluent to obtain 1.0 g of intermediate A4-6. LC-MS (ESI): [M+1]+=375.3.

[0170] Step 6: Intermediate A4-6 (1.0 g) was added to methanol (20 ml) at room temperature, followed by the addition of potassium carbonate (2.0 g) and water (5 ml). The resulting mixture was stirred for 1 to 2 hours to react. Water was added to dilute the reaction solution, which was then extracted with ethyl acetate. The resulting organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using a mixture of petroleum ether / ethyl acetate (1:0 to 2:1) as the eluent to obtain 0.65 g of intermediate A4-7. LC-MS (ESI): [M+1]+=279.3.

[0171] Step 7: Intermediate A4-7 (500 mg) was dissolved in tetrahydrofuran (20 ml) at room temperature, and pyrrole (120 mg) and tetrakis(triphenylphosphine)palladium (160 mg) were added thereto under nitrogen protection. Upon completion of the addition, the resulting mixture was stirred for 1-2 hours to react. The reaction solution was directly concentrated to dryness and purified by column chromatography using a mixture of dichloromethane / methanol (30:1) as the eluent to obtain 30 mg of compound A4 as a brown solid. LC-MS (ESI): [M+1]+=195.4.

[0172] Example 1.1.5 Synthesis of Compound A5 Compound A5 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is shown below: [ka]

[0173] Step 1: Compound A5-1 (25 g) was added to acetic acid (100 ml) at 20°C-30°C, and acetic anhydride (24.9 g) was slowly added thereto. Upon completion of the addition, the resulting mixture was stirred for 3-4 hours. The reaction solution was then slowly poured into ice water and stirred. A solid precipitated and was collected by filtration, washed with water, and dried in vacuum to give 31.0 g of intermediate A5-2 as a yellow solid. LC-MS (ESI): [M+1]+=197.2.

[0174] Step 2: Intermediate A5-2 (29 g), potassium carbonate (40 g), potassium iodide (5 g) and bromopropanol (25 g) were mixed in DMF (300 ml) at 20°C to 30°C, and the resulting mixture was heated to 100°C to 110°C and stirred for 3 to 4 hours. The reaction solution was then cooled to room temperature, quenched in ice water, and extracted three times with 2 L of ethyl acetate. The resulting organic phases were pooled, washed with saturated saline, dried over anhydrous sodium sulfate, concentrated to dryness, and then slurried with petroleum ether to obtain a solid. The solid was dried in vacuum to obtain 33.0 g of intermediate A5-3 as a yellow solid. LC-MS (ESI): [M+1]+=255.3.

[0175] Step 3: Intermediate A5-3 (23 g) was dissolved in acetonitrile (1 L) at 20°C to 25°C, and a solution of sodium dihydrogen phosphate (0.67 mol, pH 6.7, 900 ml), TEMPO (5 g), a solution of sodium chlorite (52.0 g dissolved in 60 ml of water), and a solution of sodium hypochlorite (38 ml mixed with 38 ml of water) were added thereto in sequence. Upon completion of the addition, the resulting reaction solution was dark brown and stirred for 30 minutes. Next, the reaction solution was cooled to room temperature, and 2N hydrochloric acid was added to adjust the pH to 2 to 3, and extracted with ethyl acetate (1000 ml x 3). The resulting ethyl acetate phase was pooled, washed with saturated saline, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and then slurried with petroleum ether to obtain 24.0 g of crude product of intermediate A5-4. LC-MS (ESI): [M+1]+ = 269.2.

[0176] Step 4: Intermediate A5-4 (25 g) and 5% palladium on carbon (5 g) were mixed in methanol (500 ml) at room temperature, and pressurized hydrogenation was performed for 3 to 4 hours. The palladium on carbon was filtered off, the obtained solid was washed with methanol, the filtrate was concentrated, and the obtained residue was slurried with petroleum ether to obtain a crude product. The crude product was dried in vacuum to obtain 18.0 g of intermediate A5-5 as a yellow solid. LC-MS (ESI): [M+1]+=239.5.

[0177] Then, starting from intermediate A5-5, intermediate A5-7 was synthesized according to the same procedures as those described in steps 5 and 6 of the synthesis method of compound A4, followed by deprotection with 6N hydrochloric acid to remove the acetyl group, to give compound A5 as a yellow solid. LC-MS (ESI): [M+1]+=179.2.

[0178] Example 1.1.6 Synthesis of Compound A6 Compound A6 was synthesized according to the method described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is shown below: [ka]

[0179] Compound A6 is a yellow solid. LC-MS (ESI): [M+1]+ = 248.9. 1 H NMR(400MHz,d6-DMSO):δ 12.289(1H,s), 8.666~8.648(1H,d), 8.198~8.1(1H,d), 3.138~3.108(2H,t), 2.768~2.734(2H,t), 2.219(3H,s), 2.050~1.991(2H,m)

[0180] Step 1: In a 250 mL three-necked reaction flask, intermediate A1-4 (1.25 g, 5 mmol, 1 equiv.) was dissolved in 150 ml of tetrahydrofuran under nitrogen protection. The reaction was cooled to -60°C in a dry ice bath, followed by the addition of LDA (2M in THF, 7.6 ml, 15.2 mmol, 3.04 equiv.). Upon completion of the addition, the resulting mixture was stirred at -60°C for 30 min. MeI (1.45 g, 10.21 mmol, 2.04 equiv.) was added. Upon completion of the addition, the dry ice bath was removed and the reaction was allowed to warm naturally to room temperature and maintained overnight. The next day, the reaction was quenched by the addition of 50 ml of aqueous ammonium chloride solution and extracted with ethyl acetate (150 ml x 3). The resulting organic phases were pooled, washed once with water, and concentrated to dryness to give the crude product. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (1:0 to 5:1) mixture as eluent to give 350 mg of compound A6-1 as a yellow solid in 28% yield. 1 H NMR(CDCl3):δ 12.49(1H,s), 8.77(1H,d), 8.03(1H,d), 3.21(2H,m), 2.22(3H,s), 1.90(2H,t), 1.20(6H,s)

[0181] Step 2: Compound A6-1 (280 mg) was mixed with 20 ml of 6N hydrochloric acid in a 100 ml one-neck flask, and the resulting mixture was heated under reflux and reacted for 2 hours. Heating was stopped, and the reaction solution was cooled to room temperature. Sodium bicarbonate was then added to adjust the reaction solution to pH 7-8, and the solution was extracted with dichloromethane (30 ml x 3). The resulting organic phases were pooled and concentrated to dryness to obtain 275 mg of compound A6-2 as a brownish-gray solid. The crude product was used directly in the next reaction without purification.

[0182] Step 3: Compound A6-2 (220 mg) was dissolved in acetic acid (25 ml) in a 50 ml three-neck flask, and then 1.3 g of iron powder was added to it. After the addition was completed, the resulting mixture was stirred at 80°C-85°C for 1-2 h, and when the reaction was completed, the reaction solution was distilled under reduced pressure to remove acetic acid, and water (30 ml) was added to the resulting residue. The resulting mixture was adjusted to pH 7-8 by adding sodium bicarbonate and extracted with dichloromethane (30 ml x 3). The resulting organic phase was pooled and concentrated to dryness to obtain the crude product. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (1:0 to 2:1) mixture as eluent to obtain 122 mg of compound A6 as a brown solid in 75% yield. LC-MS (ESI): [M+1]+=205.0.

[0183] Example 1.1.7 Synthesis of Compound A7 The synthetic route is shown below: [ka]

[0184] Compound A1 (2.18 g, 124 mmol, 1 equiv.) was dissolved in tetrahydrofuran (100 ml) in a 250 ml three-neck flask, and then Boc anhydride (8.2 g, 376 mmol, 3 equiv.) was added thereto. The resulting mixture was stirred at 40°C-45°C for 5 h, after which the reaction solution was directly concentrated to dryness, and the resulting residue was purified by column chromatography using a mixture of petroleum ether / ethyl acetate (1:0-1:3) as an eluent to obtain 3.0 g of compound A7-1 as a yellow solid in 90% yield. 1H NMR(400MHz,CDCl3) δ 7.25(s,1H), 6.41(t,J=10.2Hz,3H), 5.90(s,1H), 2.73(t,J=6.2Hz,2H), 2.61~2.47(m,2H), 2.02~1.86(m,3H), 1.49~1.36(m,9H)

[0185] Compound A7-1 (3.0 g, 10.8 mmol, 1 equiv.) and DIPEA (3.5 g, 27.1 mmol, 1.5 equiv.) were mixed in dichloromethane (125 ml) in a 250 ml three-neck flask. The reaction solution was cooled to -5°C to 0°C in an ice-salt bath, and acetyl chloride (1.3 g, 16.5 mmol, 2 equiv.) was added dropwise. Upon completion of the addition, the ice-salt bath was removed, and the reaction solution was allowed to warm naturally, and then reacted at room temperature with stirring for 4 h. The reaction solution was then concentrated to dryness, and the resulting residue was purified by column chromatography using a mixture of petroleum ether / ethyl acetate (1:0-1:3) as the eluent to obtain 3.48 g of compound A7-2 as a pale yellow solid in 100% yield. 1H NMR(400MHz,CDCl3) δ 12.01(s,1H), 8.55(d,J=9.1Hz,1H), 7.57(t,J=36.2Hz,1H), 6.09(s,1H), 2.80( t,J=6.2Hz,2H), 2.65~2.58(m,2H), 2.15(s,3H), 2.07~1.98(m,2H), 1.44(s,9H)

[0186] In a 500 ml three-neck flask, compound A7-2 (2.45 g, 7.7 mmol, 1 equiv) was dissolved in tetrahydrofuran (200 ml) under nitrogen protection. The reaction was cooled to -70°C to -60°C in a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 equiv) was slowly added dropwise. Upon completion of the addition, the reaction was stirred at -70°C to -60°C for 10 min. Next, a solution of NFSI in tetrahydrofuran (NFSI (7.35 g, 23 mmol, 3 equiv) dissolved in tetrahydrofuran (70 ml)) was added dropwise, and upon completion of the addition, the resulting mixture was stirred at -70°C to -60°C for 10 min. The reaction was then allowed to warm naturally to 20°C to 30°C and stirred for 3 h. The reaction was quenched by adding saturated aqueous ammonium chloride solution (80 ml), and the reaction was extracted with ethyl acetate (150 ml x 3). The resulting organic phases were pooled, washed with saturated brine, and concentrated to dryness to obtain the crude product. The resulting crude product was purified by column chromatography using a mixture of petroleum ether / ethyl acetate (1:0-1:3) as the eluent to obtain 0.85 g of compound A7-3 as a yellow solid in 31.2% yield. LC-MS (ESI): [M+1]+=355.8 1H NMR(400MHz,CDCl3) δ 11.49(s,1H), 8.64(d,J=9.2Hz,1H), 7.74(d,J=8.5Hz,1H), 6.06(s,1H), 3.01(t,J=6.4Hz,2H), 2.55~2.39(m,2H), 2.19(s,3H), 1.44(s,9H)

[0187] Compound A7-3 (0.85 g, 2.4 mmol) was mixed with 6N hydrochloric acid (30 ml) in a 100 ml one-neck flask, and the resulting mixture was heated under reflux for 4 h. Heating was then stopped, and the reaction solution was cooled to room temperature, then adjusted to pH 7-8 with sodium bicarbonate, and extracted with dichloromethane (30 ml x 3). The resulting organic phases were pooled, washed with saturated saline, and concentrated to dryness to obtain the crude product. The resulting crude product was purified by column chromatography using a petroleum ether / ethyl acetate (1:0-1:3) mixture as the eluent to obtain 0.25 g of compound A7 as a brown solid in 49.1% yield. LC-MS (ESI): [M+1]+=213.9.

[0188] Example 1.1.8 Synthesis of Compound A8 The synthetic route is shown below: [ka]

[0189] Compound A8-1 was synthesized according to the method described in Journal of Medicinal Chemistry, 1989, vol. 32, # 6, p. 1217-1230. LC-MS: (ESI): [M+1]+ = 146.3.

[0190] Compound A8-1 (1.8 g) and DIEA (4.8 g) were added to DCM (50 ml) and cooled to 0° C. Trifluoroacetic anhydride (4.8 g) was added thereto, and the resulting mixture was warmed to room temperature and reacted for 1 h. Water (20 ml) was added, the phases were separated, and the organic phase was centrifuged to dryness. The resulting residue was purified by column chromatography (EA:PE=0%-30%) to give 2.7 g of intermediate A8-2 as a white solid. LC-MS: (ESI): [M+1]+=242.2.

[0191] Zn(Et)2 (33.6 mL) was added to DCM (70 mL) and cooled to 0°C. To it was added TFA (3.8 g) and CH2I2 (9.0 g) and the resulting mixture was reacted for 15 min. A solution of compound A8-2 (2.7 g) in DCM (30 ml) was added dropwise. Upon completion of the addition, the resulting mixture was stirred at room temperature overnight. Water (30 ml) was added, the phases were separated and the organic phase was spun down to dryness to give 2.7 g of intermediate A8-3 as an off-white solid. LC-MS (ESI): [M+1]+ = 256.1.

[0192] Intermediate A8-3 (1.5 g) was added to DCM (100 ml) and acetic anhydride (30 ml) and cooled to 0° C. 65% nitric acid (2.8 g) was added thereto, and the resulting mixture was reacted at room temperature overnight. Water (50 ml) and DCM (100 ml) were added, the phases were separated, and the organic phase was spun to dryness in an oil pump to obtain 1.6 g of crude product of intermediate A8-4. The crude product was used directly in the next step. LC-MS (ESI): [M+1]+=301.2.

[0193] Intermediate A8-4 (1.6 g crude), iron powder (3.2 g) and ammonium chloride (6.4 g) were added to absolute ethanol (100 ml) and the resulting mixture was heated under reflux overnight. The reaction was cooled, filtered, spun to dryness and the resulting residue was purified by column chromatography (EA:PE=0%-50%) to give 0.25 g of intermediate A8-5 as a brown oil. LC-MS (ESI): [M+1]+=271.3.

[0194] Intermediate A8-5 (250 mg) and DIEA (400 mg) were added to DCM (10 ml) and cooled to 0° C. Trifluoroacetic anhydride (320 mg) was added thereto, and the resulting mixture was reacted at room temperature for 1 hour. The reaction solution was washed with water (5 ml) to obtain an organic phase, which was dried, filtered, and centrifuged to dryness to obtain 340 mg of intermediate A8-6 as a yellow solid. LC-MS (ESI): [M+1]+=367.3.

[0195] Intermediate A8-6 (150 mg) and CrO3 (400 mg) were added to acetic acid (8 ml) and acetic anhydride (4 ml), and the resulting mixture was reacted at 30°C to 40°C for 3 hours. The reaction solution was centrifuged to dry the solvent, and then water (5 ml) was added and extracted with DCM (20 ml). The organic phase was obtained, dried, filtered, and centrifuged to dryness to obtain 160 mg of crude product of intermediate A8-7. The crude product was used directly in the next step. LC-MS (ESI): [M+1]+ = 381.3.

[0196] Intermediate A8-7 was deprotected with 6N hydrochloric acid to remove the two trifluoroacetyl groups, and then purified by column chromatography to give compound A8. LC-MS (ESI): [M+1]+=189.3.

[0197] The following compounds can be synthesized according to similar methods as above.

[0198] [Table 1-1] [Table 1-2] [Table 1-3]

[0199] Example Group 1.2 Synthesis of Camptothecin (Friedlander Reaction) Camptothecin control compound MWC-1: [ka] was synthesized as follows:

[0200] Compound A1 (176 mg, 1 eq.), cyclic compound CDE (315 mg, 1.2 eq.) and PPTS (301 mg, 1.2 eq.) were mixed in toluene (50 ml) at room temperature, and the resulting mixture was reacted for 3 to 4 hours under reflux and water separation. The reaction solution was then directly concentrated to dryness, and the resulting residue was purified by column chromatography using a mixture of petroleum ether to methanol / dichloromethane (1:20) as the eluent, to obtain 260 mg of compound MWC-1 as a yellow solid. LC-MS (ESI): [M+1]+=404.

[0201] Example 1.2.1 Synthesis of camptothecin compound 1 The synthetic route is shown below: [ka]

[0202] In a 50 ml single-necked reaction flask, compound A6 (120 mg, 0.59 mmol, 1 equiv.), tricyclic compound CDE (180 mg, 0.68 mmol, 1.2 equiv.) and PPTS (35 mg, 0.14 mmol, 0.25 equiv.) were mixed in 15 ml under nitrogen protection, and then heated under reflux to react for 3 to 4 hours. The reaction solution was distilled under reduced pressure to remove most of the acetic acid, and the resulting residue was purified by column chromatography using a mixture of dichloromethane / methanol (15:1) as the eluent to obtain 80 mg of crude product. The crude product was further separated and purified by preparative liquid phase chromatography to obtain 3 mg of camptothecin compound 1 as a yellow solid. LC-MS(ESI):[M+1]+=432.4.

[0203] Example 1.2.2 Synthesis of Camptothecin Compound 2 (2A and 2B) The synthetic route is shown below: [ka]

[0204] Following the same method as described in Example 1.2.1, using compound A9 instead of compound A6, camptothecin compound 2A (having a short retention time) and camptothecin compound 2B (having a long retention time) were obtained after separation and purification by preparative liquid phase chromatography. Camptothecin compound 2A was obtained as a yellow solid. LC-MS (ESI): [M+1]+=418.3. Camptothecin compound 2B was obtained as a yellow solid. LC-MS (ESI): [M+1]+=418.4.

[0205] Example 1.2.3 Synthesis of camptothecin compound 3 The synthetic route is shown below: [ka]

[0206] Camptothecin compound 3 was prepared according to the same method as described in Example 1.2.1, except that compound A7 was used instead of compound A6, and toluene was used as the solvent instead of acetic acid, and obtained as an earthy yellow solid. LC-MS (ESI): [M+1]+=440.8. 1H NMR (400MHz, acetone) δ 7.89(d,J=9.1Hz,1H), 7.48(d,J=9.1Hz,1H), 7.37(s,1H), 7.31(d,J=8.9Hz,0H), 5.59~5.26( m,5H), 3.11~3.05(m,4H), 2.73~2.59(m,3H), 1.98(dt,J=14.0,7.0Hz,1H), 1.05~0.98(m,3H) 1H NMR(400MHz,DMSO) δ 7.87(d,J=9.1Hz,0H), 7.41(d,J=9.1Hz,0H), 7.21(s,0H), 6.50(s,0H), 6.15(s,1H), 5.36(d,J=46.6H) z,1H), 2.93(t,J=6.5Hz,1H), 2.66~2.54(m,1H), 1.97~1.68(m,3H), 1.24(s,1H), 0.88(t,J=7.3Hz,3H)

[0207] Example 1.2.4 Synthesis of camptothecin compound 4 (4A and 4B) [ka] Following the same method as described in Example 1.2.1, using compound A10 instead of compound A6, camptothecin compound 4A (having a short retention time) and camptothecin compound 4B (having a long retention time) were obtained after separation and purification by preparative liquid phase chromatography. Compound 4A was obtained as an ocher solid. LC-MS (ESI): [M+1]+=422.3. Camptothecin compound 4B was obtained as an ocher solid. LC-MS (ESI): [M+1]+=422.3.

[0208] Example 1.2.5 Synthesis of camptothecin compound 5 The synthetic route is shown below: [ka]

[0209] Camptothecin compound 5 was prepared according to a similar method as described in Example 1.2.1, using compound A12 instead of compound A6, and obtained as a yellow solid. LC-MS (ESI): [M+1]+=444.3.

[0210] Example 1.2.6 Synthesis of camptothecin compound 6 The synthetic route is shown below: [ka]

[0211] Camptothecin compound 6 was prepared according to a similar method as described in Example 1.2.1, using compound A11 instead of compound A6, and obtained as a yellow solid. LC-MS (ESI): [M+1]+=430.5.

[0212] Example 1.2.7 Synthesis of camptothecin compound 7 The synthetic route is shown below: [ka]

[0213] Following a similar method to that described in Example 1.2.1, compound A8 was used instead of compound A6, 0.7 mg of camptothecin compound 7 was prepared as a yellow solid. LC-MS (ESI): [M+1]+=416.3.

[0214] Example 1.2.8 Synthesis of camptothecin compound 9 The synthetic route is shown below: [ka]

[0215] Following a similar method to that described in Example 1.2.1, but using compound A3 instead of compound A6, camptothecin compound 9 was prepared and obtained as a brownish red solid. LC-MS (ESI): [M+1]+=420.3.

[0216] Example 1.2.9 Synthesis of camptothecin compound 12 Compound 12 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is shown below: [ka]

[0217] Following the same method as described in Example 1.2.1, using compound A4 instead of compound A6, and using toluene as the solvent instead of acetic acid, 30 mg of camptothecin compound 12 was synthesized and obtained as a brown solid. LC-MS (ESI): [M+1]+=422.5.

[0218] Example 1.2.10 Synthesis of camptothecin compound 13 The synthetic route is shown below: [ka]

[0219] Compound 13 was synthesized according to a similar method as described in Example 1.2.1, except that compound A15 was used instead of compound A6 and toluene was used as the solvent, and was obtained as a brown solid. LC-MS (ESI): [M+1]+=458.4.

[0220] Example 1.2.11 Synthesis of camptothecin compound 14 The synthetic route is shown below: [ka]

[0221] Compound 14 was synthesized according to a similar method as described in Example 1.2.1, except that compound A5 was used instead of compound A6 and toluene was used as the solvent, and obtained as a yellow solid. LC-MS (ESI): [M+1]+=406.1.

[0222] Example 1.2.12 Synthesis of camptothecin compound 14-P [ka] Compound 12 (200 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml) at room temperature, and the resulting mixture was cooled to 5°C under nitrogen protection, and then 30% H2O2 (580 mg) was added thereto. The reaction solution was stirred for 2 hours, concentrated to dryness, and the resulting residue was subjected to preparative HPLC to obtain compounds 14-P1 and 14-P2 separately. After lyophilization, 42 mg of compound 14-P1 was obtained as a brown solid, and 25 mg of compound 14-P2 was obtained as a brown solid. LC-MS (ESI): [M+1]+ = 438.5.

[0223] Example 1.2.13 Synthesis of camptothecin compound 15 [ka] Compound 12 (100 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml) at room temperature, and the resulting mixture was cooled to 5° C. under nitrogen protection, and then 30% H2O2 (750 mg) was added thereto. The reaction solution was stirred for 2 hours, warmed to room temperature (25° C.), and stirred overnight. After completion of the reaction as confirmed by LCMS analysis, the reaction solution was concentrated to dryness, and the resulting residue was subjected to preparative HPLC to separate and recover compound 15. Lyophilization gave 8 mg of compound 15 as a brown solid. LC-MS (ESI): [M+1]+=454.4.

[0224] Example 1.2.14 Synthesis of camptothecin compound 16 [ka] Intermediate 16a was synthesized according to the method described in WO2020200880, and then intermediate 16c having the structure of 16a-thiocamptothecin was prepared according to the method described in the literature (J. Med. Chem. 2008, 51, 3040-3044), and finally deprotected to remove the acetyl group to prepare compound 16. LC-MS (ESI): [M+1]+=420.3.

[0225] Example 1.2.15 Synthesis of camptothecin compound 17 The synthetic route is shown below: [ka]

[0226] Following the same method as described in Example 1.2.1, using compound A1 instead of compound A6, HCDE instead of CDE, and toluene as the solvent, homocamptothecin compound 17 (mixture of S and R configurations) was obtained. LC-MS (ESI): [M+1]+=418.5.

[0227] Example 1.2.16 Synthesis of camptothecin compound 18 The synthetic route is shown below: [ka]

[0228] Following a similar method to that described in Example 1.2.1, using compound A16 instead of compound A6 and toluene as the solvent, compound 18 was synthesized as a pale yellow solid. LC-MS (ESI): [M+1]+=424.2.

[0229] Example 1.2.17 Synthesis of camptothecin compound 19 The synthetic route is shown below: [ka]

[0230] Following a similar method to that described in Example 1.2.1, compound A17 was used instead of compound A6, and toluene was used as the solvent, to synthesize compound 19 as a yellow solid. LC-MS (ESI): [M+1]+=460.5.

[0231] Example 1.2.18 Synthesis of camptothecin compound 20 The synthetic route is shown below: [ka]

[0232] Following a similar method to that described in Example 1.2.1, compound A13 was used instead of compound A6, and toluene was used as the solvent, to synthesize compound 20 as a yellow solid. LC-MS (ESI): [M+1]+=458.2.

[0233] Example 1.2.19 Synthesis of camptothecin compound 21 The synthetic route is shown below: [ka]

[0234] Following a similar method to that described in Example 1.2.1, using compound A24 instead of compound A6 and toluene as the solvent, compound 21 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=418.4.

[0235] Example 1.2.20 Synthesis of camptothecin compound 22 The synthetic route is shown below: [ka]

[0236] Following a similar method to that described in Example 1.2.1, using compound A25 instead of compound A6 and toluene as the solvent, compound 22 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=434.4.

[0237] Example 1.2.21 Synthesis of camptothecin compound 23 The synthetic route is shown below: [ka]

[0238] Following a similar method to that described in Example 1.2.1, using compound A21 instead of compound A6 and toluene as the solvent, compound 23 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=432.4.

[0239] Example 1.2.22 Synthesis of camptothecin compound 24 The synthetic route is shown below: [ka]

[0240] Following a similar method to that described in Example 1.2.1, using compound A20 instead of compound A6 and toluene as the solvent, compound 24 (isomer pair) was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=418.4.

[0241] Example 1.2.23 Synthesis of camptothecin compound 25 The synthetic route is shown below: [ka]

[0242] Following a similar method to that described in Example 1.2.1, using compound A32 instead of compound A6 and toluene as the solvent, compound 25 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=432.4.

[0243] Example 1.2.24 Synthesis of camptothecin compound 26 [ka] Following a similar method to that described in Example 1.2.1, but using compound A18 instead of compound A6 and toluene as the solvent, compound 26 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=440.4.

[0244] Example 1.2.25 Synthesis of camptothecin compound 27 [ka] Following a similar method to that described in Example 1.2.1, using compound A19 instead of compound A6 and toluene as the solvent, compound 27 was obtained as a brownish red solid. LC-MS (ESI): [M+1]+=476.4.

[0245] Example 1.2.26 Synthesis of camptothecin compound 31 [ka] Following a similar method to that described in Example 1.2.1, but using compound A14 instead of compound A6 and toluene as the solvent, compound 31 was obtained as a yellow solid. LC-MS (ESI): [M+1]+=442.4.

[0246] Example 1.2.27 Synthesis of camptothecin compound 32 [ka] Compound 32 was synthesized according to a similar method to that described in Example 1.2.1, except that compound A2 was used instead of compound A6, HCDE was used instead of CDE, and toluene was used as the solvent, and obtained as a yellow solid. LC-MS (ESI): [M+1]+=436.4.

[0247] Example 1.2.28 Synthesis of camptothecin compound 33 The synthetic route is shown below: [ka]

[0248] Following a similar method to that described in Example 1.2.1, compound A23 was used instead of compound A6, and toluene was used as the solvent, to synthesize compound 33 as a yellow solid. LC-MS (ESI): [M+1]+=458.5.

[0249] Example 1.2.29 Synthesis of camptothecin compound 34 The synthetic route is shown below: [ka]

[0250] Following a similar method to that described in Example 1.2.1, compound 34 was synthesized as a yellow solid using compound A22 instead of compound A6 and toluene as the solvent. LC-MS (ESI): [M+1]+=458.5.

[0251] Example 1.2.30 Synthesis of camptothecin compound 35 and compound 38 The synthetic route is shown below: [ka]

[0252] Following a similar method to that described in Example 1.2.1, using compound A26 instead of compound A6 and toluene as the solvent, compound 35 was synthesized as a brownish red solid. LC-MS (ESI): [M+1]+=438.7.

[0253] Compound 38 was synthesized according to a similar method as described in Example 1.2.1, except that compound A27 was used instead of compound A6 and toluene was used as the solvent, and was obtained as a brownish-red solid. LC-MS (ESI): [M+1]+=474.7.

[0254] Example 1.2.31 Synthesis of camptothecin compound 36 and compound 39 The synthetic route is shown below: [ka]

[0255] Following a similar method to that described in Example 1.2.1, using compound A30 instead of compound A6 and toluene as the solvent, compound 36 was synthesized as a brownish red solid. LC-MS (ESI): [M+1]+=456.7.

[0256] Compound 39 was synthesized according to a similar method as described in Example 1.2.1, except that compound A31 was used instead of compound A6 and toluene was used as the solvent, and was obtained as a brownish-red solid. LC-MS (ESI): [M+1]+=492.7.

[0257] Example 1.2.32 Synthesis of camptothecin compound 37 and compound 40 The synthetic route is shown below: [ka]

[0258] Following a similar method to that described in Example 1.2.1, using compound A28 instead of compound A6 and toluene as the solvent, compound 37 was synthesized as a brownish red solid. LC-MS (ESI): [M+1]+=440.8.

[0259] Compound 40 was synthesized according to a similar method as described in Example 1.2.1, except that compound A29 was used instead of compound A6 and toluene was used as the solvent, and was obtained as a brownish-red solid. LC-MS (ESI): [M+1]+=476.7.

[0260] Example Group 2: Synthesis of Drug-Containing Linkers Example 2.1 Synthesis of Drug-Containing Linker MWD-L1 [ka] The synthetic route is shown below: [ka]

[0261] GGFG-Dxd was synthesized according to the method described in US Patent Publication No. 20190151328: [ka]

[0262] GGFG-Dxd was obtained as a pale yellow solid. LC-MS (ESI): M+1=841.

[0263] The linker compound BL was synthesized according to the method described in WO 2018 / 095422: [ka]

[0264] Linker compound BL was obtained as a yellow solid. LC-MS (ESI): M+1=858.

[0265] Linker compound BL (857 mg, 1 mmol), GGFG-Dxd (840 mg, 1 mmol, 1 equiv), DIPEA (323 mg, 2.5 mmol, 2.5 equiv) and HATU (570 mg, 1.5 mmol, 1.5 equiv) were dissolved in 30 ml of DCM and reacted with stirring for 2 h. The reaction solution was cooled to 5°C-10°C, 1N hydrochloric acid (20 ml) was added to it, and then stirred for 0.5 h. Separated phases were obtained and the aqueous phase was extracted with DCM (30 ml x 2). The obtained organic phases were then pooled, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated and centrifuged to dryness. The obtained residue was purified by column chromatography using DCM / MeOH (50:1-10:1) mixture as eluent to obtain 500 mg of drug-containing linker MWD-L1 as a yellow solid in 29.8% yield. LC-MS (ESI): M+1=1681,(M+1) / 2=840.9.

[0266] Example 2.2 Synthesis of Drug-Containing Linker MWC-L2 [ka]

[0267] Step 1: Boc-Val-Ala-OH (288 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 equiv.), DIPEA (322 mg, 2.5 mmol, 2.5 equiv.) and HATU (456 mg, 1.2 mmol, 1.2 equiv.) were dissolved in 30 ml of DCM and reacted with stirring for 2 hours. The reaction solution was centrifuged to dryness, and the resulting residue was purified by column chromatography using a mixture of PE / EA (10:1-5:1) as an eluent to quantitatively obtain 450 mg of intermediate 2-1 as a gray-green solid.

[0268] Step 2: Intermediate 2-1 (450 mg, 1 mmol), tricyclic CDE (263 mg, 1 mmol, 1 equiv.) and pyridinium p-toluenesulfonate (PPTS) (251 mg, 1 mmol, 1 equiv.) were suspended in 30 ml of toluene and heated under reflux for 2 h. Heating was stopped, the reaction was cooled, and the solid precipitate was collected to give 750 mg of crude intermediate 2-2 as a brown solid. LC-MS (ESI): M+1=574. The crude product was used directly in the next step without purification.

[0269] Step 3: Linker compound BL (857 mg, 1 mmol), HoSu (138 mg, 1.2 mmol, 1.2 eq.) and DCC (310 mg, 1.5 mmol, 1.5 eq.) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was aspirated, and the obtained filtrate was a solution of A-Osu in DCM. The filtrate was added to a mixed solution of crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq.) and DCM (30 ml), which was then stirred for 3 h to react. The reaction solution was then cooled to below 10° C., and 1N hydrochloric acid (20 ml) was added thereto, followed by stirring for 0.5 h. Separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml×2). The obtained organic phases were then pooled, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dryness. The resulting residue was purified by column chromatography using a mixture of DCM / MeOH (50:1-10:1) as the eluent to give 325 mg of the drug-containing linker MWC-L2 as an orange solid in 23.0% yield. LC-MS (ESI): M+1=1414, (M+1) / 2=707.

[0270] Example 2.3 Synthesis of Drug-Containing Linker MWC-L3 [ka] Step 1:

[0271] Boc-Gly-OH (175 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 equiv.), DIPEA (322 mg, 2.5 mmol, 2.5 equiv.) and HATU (456 mg, 1.2 mmol, 1.2 equiv.) were dissolved in 30 ml of DCM and reacted with stirring for 2 h. The reaction solution was centrifuged to dryness, and the resulting residue was purified by column chromatography using a mixture of PE / EA (10:1-5:1) as an eluent to quantitatively obtain 335 mg of intermediate 3-1 as a light brown solid.

[0272] Step 2: Intermediate 3-1 (335 mg, 1 mmol), tricyclic CDE (263 mg, 1 mmol, 1 equiv.) and PPTS (251 mg, 1 mmol, 1 equiv.) were suspended in 30 ml of toluene and heated under reflux for 2 h. Heating was stopped, the reaction was cooled, and the solid precipitate was collected to give 560 mg of crude intermediate 3-2 as a brown solid. LC-MS (ESI): M+1=461. The crude product was used directly in the next step without purification.

[0273] Step 3: Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq.) and HATU (456 mg, 1.2 mmol, 1.2 eq.) were dissolved in 20 ml of DCM and reacted with stirring for 3 h. 1N hydrochloric acid (30 ml) was added thereto, followed by stirring for 0.5 h. The reaction solution was extracted with DCM (30 ml x 2). The resulting organic phases were then pooled, washed with saturated brine, dried over anhydrous sodium sulfate, pumped and centrifuged to dryness. The resulting residue was purified by column chromatography using a mixture of DCM / MeOH (50:1-10:1) as the eluent to give 350 mg of intermediate 3-3 as a brown solid. LC-MS (ESI): M+1=945.

[0274] Step 4: Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), and then diethylamine (2 ml) was added thereto, and the resulting mixture was stirred for 3 hours to react. The reaction solution was centrifuged to dryness to obtain the crude product of intermediate 3-4 as a brown sticky substance. LC-M (ESI): M+1=722. The crude product was used directly in the next step without purification.

[0275] Step 5: Linker compound BL (387 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq.) and DCC (114 mg, 0.56 mmol, 1.5 eq.) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was aspirated and the obtained filtrate was added to a mixed solution of crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq.) and DCM (30 ml), which was then stirred for 3 h to react. The reaction solution was then cooled to below 10° C., 1N hydrochloric acid (20 ml) was added thereto, followed by stirring for 0.5 h. Separated phases were obtained and the aqueous phase was extracted with DCM (30 ml×2). The obtained organic phases were then pooled, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dryness. The resulting residue was purified by column chromatography using a mixture of DCM / MeOH (50:1-10:1) as the eluent to give 120 mg of the drug-containing linker MWC-L3 as an orange solid in 20.8% yield. LC-MS (ESI): M+1=1562, (M+1) / 2=781.

[0276] Example 2.4 Synthesis of Drug-Containing Linker MWE-L4 [ka] MWE-L4 was synthesized according to the method described in Example 2.2, using compound A3 instead of compound A1, and obtained as an orange solid. LC-MS (ESI): M+1=1430.

[0277] Example 2.5 Synthesis of Drug-Containing Linker MWG-L5 [ka] MWG-L5 was synthesized according to the method described in Example 2.2, using compound A6 instead of compound A1, and obtained as an orange solid. LC-MS (ESI): M+1=1442.

[0278] Example 2.6 Synthesis of drug-containing linker MWF-L6 [ka] MWF-L6 was synthesized according to the method described in Example 2.2, using compound A7 instead of compound A1, and obtained as an orange solid. LC-MS (ESI): M+1=1450.

[0279] Example 2.7 Synthesis of drug-containing linker MWF-L7 [ka] MWF-L7 was synthesized according to the method described in Example 2.2, except that compound A7 was used instead of compound A1 and Alloc-Ala-Ala-Ala-OH was used instead of Alloc-Val-Ala-OH, and was obtained as an orange solid. LC-MS (ESI): M+1=1493.5.

[0280] Example 2.8 Synthesis of drug-containing linker MWF-L8 [ka] MWF-L8 was synthesized according to the method described in Example 2.3, using compound A7 instead of compound A1, and obtained as an orange solid. LC-MS (ESI): M+1=1598.4.

[0281] Example 2.9 Synthesis of drug-containing linker MWF-L9 [ka] MWF-L9 was synthesized according to the method described in Example 2.2, using compound A5 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1416.4

[0282] Example 2.10 Synthesis of drug-containing linker MWF-L10 [ka] MWF-L10 was synthesized according to the method described in Example 2.2, using compound A13 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1468.4.

[0283] Example 2.11 Synthesis of drug-containing linker MWF-L11 [ka] MWF-L11 was synthesized according to the method described in Example 2.2, using compound A18 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1450.4.

[0284] Example 2.12 Synthesis of drug-containing linker MWF-L12 [ka] MWF-L12 was synthesized according to the method described in Example 2.2, using compound A16 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1434.4.

[0285] Example 2.13 Synthesis of drug-containing linker MWF-L13 [ka] MWF-L13 was synthesized according to the method described in Example 2.2, using compound A19 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1486.4.

[0286] Example 2.14 Synthesis of drug-containing linker MWF-L14 [ka] MWF-L14 was synthesized according to the method described in Example 2.2, using compound A17 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1470.4.

[0287] Example 2.15 Synthesis of drug-containing linker MWF-L15 [ka] MWF-L15 was synthesized according to the method described in Example 2.2, using compound A2 instead of compound A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1432.4.

[0288] Example 2.16 Synthesis of Drug-Containing Linker MWD-L7 [ka] The synthetic route is as follows: [ka]

[0289] Step 1: Fmoc-Val-Cit-PAB-PNP (153 mg, 0.2 mmol), exatecan mesylate (106 mg, 0.2 mmol, 1 eq.) and DIPEA (65 mg, 0.5 mmol, 2.5 eq.) were dissolved in 30 ml of DCM and reacted with stirring for 2 h. The reaction solution was centrifuged to dryness, and the resulting residue was purified by column chromatography using a mixture of DCM / MeOH (50:1-10:1) as the eluent to obtain 180 mg of intermediate 5-1 as a yellow solid. LC-MS (ESI): M+1=1064.

[0290] Step 2: Intermediate 5-1 (180 mg, 0.17 mmol) was dissolved in methanol (20 ml), and then diethylamine (2 ml) was added thereto, and the resulting mixture was stirred for 3 hours to react. The reaction solution was centrifuged to dryness to obtain the crude product of intermediate 5-2 as a pale yellow sticky substance. LC-M (ESI): M+1 = 842. The crude product was used directly in the next step without purification.

[0291] Step 3: Linker compound BL (146 mg, 0.17 mmol), HoSu (25 mg, 0.21 mmol, 1.2 eq.) and DCC (54 mg, 0.26 mmol, 1.5 eq.) were dissolved in 20 ml of DCM and stirred at room temperature for 3 h. The reaction solution was aspirated, and the obtained filtrate was added to a mixed solution of crude intermediate 5-1, DIPEA (56 mg, 0.43 mmol, 2.5 eq.) and DCM (20 ml), which was then stirred for 3 h to react. The reaction solution was then cooled to 5°C to 10°C, 1N hydrochloric acid (20 ml) was added thereto, followed by stirring for 0.5 h. Separated phases were obtained, and the aqueous phase was extracted with DCM (30 ml x 2). The obtained organic phases were then pooled, washed with saturated brine, dried over anhydrous sodium sulfate, aspirated, and centrifuged to dryness. The resulting residue was purified by column chromatography using a mixture of DCM / MeOH (50:1-10:1) as the eluent to give 67 mg of the drug-containing linker MWD-L7 as a yellow solid in 23.1% yield. LC-MS (ESI): M+1=1682, (M+1) / 2=841.

[0292] Example 2.17 Synthesis of Drug-Containing Linker MWD-L8 [ka] Instead of compound BL, [ka] MWD-L8 was synthesized according to a similar method as described in Example 2.1 using and obtained as a pale yellow solid. LC-MS (ESI): M+1=1645.7.

[0293] Example 2.18 Synthesis of Drug-Containing Linker MWD-L9 [ka] Instead of compound BL, [ka] MWD-L9 was synthesized according to a similar method as described in Example 2.1 using the compound obtained as a pale yellow solid. LC-MS (ESI): M+1=1713.7.

[0294] Example 2.19 Synthesis of drug-containing linker LD [ka] LD was synthesized following a similar method as described in Example 2.5 using MC-OSU instead of BL-OSU, and obtained as a pale yellow solid. LC-MS (ESI): M+1=795.9.

[0295] Example 2.20 Synthesis of drug-containing linker LE [ka] LE was synthesized following a similar method as described in Example 2.2, using MC-OSU instead of BL-OSU, and obtained as a pale yellow solid. LC-MS (ESI): M+1=803.8.

[0296] Example 2.21 Synthesis of drug-containing linker LF [ka] LF was prepared according to a similar method as described in Example 2.2, using A7 instead of A1 and MaL-PEG8-COOH instead of compound BL, and obtained as a yellow solid. LC-MS (ESI): M+1=1185.

[0297] Example 2.22 Synthesis of drug-containing linker LG [ka] LG was prepared according to a similar method as described in Example 2.3 using compound Mc-OSu instead of compound BL-OSu, and obtained as a yellow solid. LC-MS (ESI): M+1=965.98.

[0298] Example 2.23 Synthesis of drug-containing linker LH [ka] Following a similar method as described in Example 2.4, using MaL-PEG8-COOH instead of compound BL, LH was prepared and obtained as a pale yellow solid. LC-MS (ESI): M+1=1165.3.

[0299] Example 2.24 Synthesis of Drug-Containing Linker LI [ka] LI was prepared following a similar method as described in Example 2.2 using A13 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1203.4.

[0300] Example 2.25 Synthesis of Drug-Containing Linker LJ [ka] LJ was prepared following a similar method as described in Example 2.2 using A18 instead of A1, and was obtained as a yellow solid. LC-MS (ESI): M+1=1185.4.

[0301] Example 2.26 Synthesis of Drug-Containing Linker LK [ka] LK was prepared following a similar method as described in Example 2.2 using A16 instead of A1, and was obtained as a yellow solid. LC-MS (ESI): M+1=1169.3.

[0302] Example 2.27 Synthesis of Drug-Containing Linker LL [ka] LL was prepared following a similar method as described in Example 2.2 using A19 instead of A1, and was obtained as a yellow solid. LC-MS (ESI): M+1=1221.4.

[0303] Example 2.28 Synthesis of drug-containing linker LM [ka] LM was prepared following a similar method as described in Example 2.2 using A17 instead of A1, and was obtained as a yellow solid. LC-MS (ESI): M+1=1205.4.

[0304] Example 2.29 Synthesis of drug-containing linker LN [ka] Following the same method as described in Example 2.2, using intermediate HCDE instead of intermediate CDE and using MaL-PEG8-COOH instead of compound BL, LN was prepared as a yellow solid. LC-MS (ESI): M+1=1163.2 (mixture of diastereoisomeric pair).

[0305] Example 2.30 Synthesis of Drug-Containing Linker MWS-L1 [ka] Instead of A1, A13 is used, and instead of the linker compound BL, [ka] MWS-L1 was prepared according to a similar method as described in Example 2.2 using and obtained as a yellow solid. LC-MS (ESI): M+1=1236.3.

[0306] Example 2.31 Synthesis of Drug-Containing Linker MWS-L2 [ka] MWS-L2 was prepared following a similar method as described in Example 2.2 using A18 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1218.4.

[0307] Example 2.32 Synthesis of drug-containing linker MWS-L3 [ka] MWS-L3 was prepared following a similar method as described in Example 2.2 using A16 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1202.4.

[0308] Example 2.33 Synthesis of drug-containing linker MWS-L4 [ka] MWS-L4 was prepared following a similar method as described in Example 2.2 using A19 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1254.4.

[0309] Example 2.34 Synthesis of Drug-Containing Linker MWS-L5 [ka] MWS-L5 was prepared following a similar method as described in Example 2.2 using A17 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1238.4.

[0310] Example 2.35 Synthesis of Drug-Containing Linker MWS-L6 [ka] Instead of Mal-PEG8-COOH, [ka] MWS-L6 was prepared following a similar method as described in Example 2.11 using and obtained as a yellow solid. LC-MS (ESI): M+1=1302.4.

[0311] Example 2.36 Synthesis of drug-containing linker MWS-L7 [ka] Following a similar method as described in Example 2.2, using A18 instead of A1, MWS-L7 was prepared and obtained as a yellow solid. LC-MS (ESI): M+1=1284.4.

[0312] Example 2.37 Synthesis of Drug-Containing Linker MWS-L8 [ka] Following a similar method as described in Example 2.2, using A16 instead of A1, MWS-L8 was prepared and obtained as a yellow solid. LC-MS (ESI): M+1=1268.4.

[0313] Example 2.38 Synthesis of Drug-Containing Linker MWS-L9 [ka] MWS-L9 was prepared following a similar method as described in Example 2.2 using A19 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1320.4.

[0314] Example 2.39 Synthesis of drug-containing linker MWS-L10 [ka] MWS-L10 was prepared following a similar method as described in Example 2.2 using A17 instead of A1, and obtained as a yellow solid. LC-MS (ESI): M+1=1304.4.

[0315] Example Group 3: Synthesis of Comparative Drug-Containing Linkers Example 3.1 Synthesis of drug-containing linkers LA, LB, and LC 3.1.1 Synthesis of drug-containing linkers for stochastic conjugation The drug-containing linker MC-GGFG-Dxd was synthesized according to the method described in US Patent Publication No. 20190151328, and MC-GGFG-Dxd was obtained as a pale yellow solid. LC-MS (ESI): M+1=1034. 1 H NMR (CDCl3).

[0316] The drug-containing linkers LA and LB were synthesized according to the method described in WO2020200880, and LA was obtained as a yellow solid (LC-MS(ESI):M+1=767) and LB was obtained as a brown sticky material (LC-MS(ESI):M+1=1149). [ka]

[0317] 3.2.2 Synthesis of other drug-containing linkers LC for ring bridging [ka] The synthetic route is as follows: [ka]

[0318] Following a similar method to that of drug-containing linker L-1, linker LC was synthesized as a pale yellow solid. LC-MS (ESI): M+1=1982, (M+1) / 2=991.

[0319] Group of Examples 4 Preparation and Physicochemical Characterization of Antibody-Drug Conjugates The process for preparing the antibody-drug conjugate is as follows.

[0320] a. Site-specific conjugation process [ka] The antibody is reduced to cleave disulfide bonds, the reduced antibody is conjugated with a linker to form a cross-linked antibody-drug conjugate, followed by hydrolysis to open the maleimide ring and purification of the resulting product to yield the antibody-drug conjugate of DAR 4.

[0321] b. Stochastic conjugation process [ka] The antibody was reduced to cleave disulfide bonds and then conjugated with a linker to form a cross-linked antibody-drug conjugate.

[0322] An exemplary experimental method used is as follows.

[0323] 4.1 Common methods for preparing antibody-drug conjugates a. Common preparation method for site-specific conjugation Antibody reduction: A sample containing 120 mg of antibody is transferred into a buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.0) using a NAP-25 chromatography column packed with Sephadex G-25, and the antibody concentration is diluted to 10 mg / mL in the buffer solution. 2.1 ml of an aqueous solution of 10 mg / mL TCEP (Sigma-Aldrich) is added to 10 mL of diluted antibody sample (total 100 mg) at an equivalent molar ratio of 1:10 (antibody:TCEP). After 2 hours of incubation, the reaction solution is subjected to buffer exchange into a buffer solution containing 50 mM NaCl and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 6.5) using a Sephadex G-25 chromatography column.

[0324] Conjugation and hydrolysis of antibody and drug-containing linker: The above reduced antibody is diluted to 5 mg / mL, and 0.38 mL of N,N-dimethylacetamide (DMA) accounting for 2% of the total reaction volume as a presolvent and a solution of drug-containing linker in DMA at 10 mg / mL are added sequentially in an equivalent molar ratio of 1:5.5 (antibody: drug-containing linker) as a reaction solution. The resulting mixture is stirred at room temperature for 30 minutes and then subjected to buffer exchange into a buffer consisting of disodium hydrogen phosphate-sodium dihydrogen phosphate (pH 8.0) using a NAP-25 chromatography column packed with Sephadex G-25 to remove excess drug-containing linker. The resulting solution is heated in a water bath at 37 °C for 3 hours.

[0325] Purification of antibody-drug conjugate: A sample of the above solution is concentrated to a concentration of about 15 mg / mL using an AMICOM ultrafiltration centrifuge tube. A buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate and 3 M ammonium phosphate is added to achieve a conductivity of 100 ms / cm. The solution is then applied to a hydrophobic column packed with TOYOPEARL Butyl-650M (purchased from Tosoh Corporation) using phase A: a buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate and 0.6 M ammonium sulfate; phase B: a buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate. Eight column volume gradient elutions are performed with 0% to 100% phase B to collect the main peak.

[0326] The final sample obtained was subjected to buffer exchange into a buffer consisting of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate (pH 7.4) using AMICOM ultrafiltration centrifuge tubes, and then filtered through a 0.22 μm filter (stedim Minisart from Sartorius).

[0327] b. Common preparation method for stochastic conjugation The stochastically conjugated antibody-drug conjugate is prepared and obtained according to the preparation method described in Chinese Patent Publication No. 105849126.

[0328] 4.2 Common methods for analyzing antibody-drug conjugates a. Ultraviolet spectrophotometry for determining drug-antibody ratio (UV-DAR method) and concentration The concentration of the antibody-drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the absorption wavelength characteristic of the small molecule and calculating as follows:

[0329] a1. Determination of Drug-Antibody Ratio (DAR) value of antibody-drug conjugate From the literature (Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70), DAR (drug-antibody ratio) = (ε Ab 280 -A 280 / A z ×ε Ab z ) / (A 280 / A z ×ε D z -ε D 280 ), where ε Ab 280 is the molar absorption coefficient of the antibody at 280 nm, and A 280 is the UV absorbance of the antibody-drug conjugate at 280 nm, A z is the UV absorbance of the antibody-drug conjugate at Zn, the absorption wavelength characteristic of the drug-containing linker in the antibody-drug conjugate, and ε Ab z is the molar absorption coefficient of the antibody at the absorption wavelength characteristic of the drug-containing linker, Znm, and ε D z is the molar absorption coefficient of the drug-containing linker at Znm, and ε D 280 is the molar absorption coefficient of the drug-containing linker at 280 nm, as follows:

[0330] [Table 2]

[0331] a2. Determining the concentration of antibody-drug conjugate Because the total absorbance of a system at a particular wavelength is equal to the sum of the absorbances of all absorbing species present in the system (additivity of absorbance), if the molar absorption coefficients of the antibody and drug-containing linker involved do not change before and after conjugation of the antibody to the drug-containing linker, the concentration of the antibody-drug conjugate is determined by the relationship: A 280 =ε ADC 280 ×C ADC ×L=(ε D 280 ×DAR+ε Ab 280 ) C ADC × L.

[0332] Therefore, the molar concentration (mol / L) of the antibody-drug conjugate is C ADC =A 280 / (ε Ab 280 +ε D 280 × DAR). Therefore, the concentration of the antibody-drug conjugate (g / L) is C ADC =A 280 / (ε Ab 280 +ε D 280 ×DAR)×MW ADC =A 280 / (ε Ab 280 +ε D 280 ×DAR)×(MW Ab +MW D × DAR), where MW ADC is the molecular weight of the antibody-drug conjugate, and MW Ab is the molecular weight of the antibody, and MW D is the molecular weight of the drug-containing linker, and the protein concentration can be obtained by inserting the DAR value into the equation.

[0333] B Hydrophobic chromatography b1. Hydrophobic chromatography (HIC-HPLC) for determining the DAR value of antibody-drug conjugates Sample preparation: The sample is diluted to 2.0 mg / mL with mobile phase B, then centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant is subjected to HPLC analysis. Chromatography column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm × 35 mm; Mobile phase A: 1.5M(NH4)2SO4+25mM PB, pH7.0; Mobile phase B: 25mM PB+20%IPA, pH7.0; Flow rate: 0.6mL / min; Detection wavelength: 280nm; Column temperature: 30°C; Loading volume: 10 μL; Chromatographic gradient used for HIC analysis:

[0334] [Table 3]

[0335] Formula for calculating DAR: DAR=Σ(weighted peak area) / 100, i.e., DAR=(D0 peak area ratio×0+D1 peak area ratio×1+D2 peak area ratio×2+D3 peak area ratio×3+D4 peak area ratio×4+D5 peak area ratio×5+D6 peak area ratio×6+D7 peak area ratio×7+D8 peak area ratio×8) / 100.

[0336] b2. Hydrophobic chromatography (HIC-HPLC) for determining the DAR value of antibody-drug conjugates Sample preparation: The sample is diluted to 2.0 mg / mL with mobile phase B, then centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant is subjected to HPLC analysis. Chromatography column: TSKgel Butyl-NPR, 2.5 μm, 4.6 mm × 100 mm; Mobile phase A: 1.2M(NH4)2SO4+25mM PB, pH7.0; Mobile phase B: 25mM PB+20%IPA, pH7.0; Flow rate: 0.6mL / min; Detection wavelength: 280nm; Column temperature: 30°C; Loading volume: 10 μL; Chromatographic gradient used for HIC analysis:

[0337] [Table 4]

[0338] DAR calculation formula: Same as b1.

[0339] b3. Hydrophobic chromatography (HIC-HPLC) for determining the DAR value of antibody-drug conjugates Sample preparation: The sample is diluted to 2.0 mg / mL with mobile phase B, then centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant is subjected to HPLC analysis. Chromatography column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm × 35 mm; Mobile phase A: 2.5M(NH4)2SO4+125mM PB, pH7.0; Mobile phase B: 125mM PB, pH7.0; Mobile phase C: IPA; Mobile phase D: H2O; Flow rate: 0.5mL / min; Detection wavelength: 280nm; Column temperature: 30°C; Loading volume: 10 μL; Chromatographic gradient used for HIC analysis:

[0340] [Table 5]

[0341] DAR calculation formula: Same as b1.

[0342] c. Mass spectrometry (LC-MS) used to determine DAR value Sample processing: An appropriate amount of sample is placed in an ultrafiltration tube and subjected to buffer exchange into a buffer consisting of 50 mM NH4HCO3 (pH 7.1). After refilling the buffer, the resulting sample is subjected to ultrafiltration centrifugation (13000g x 5 min). After buffer exchange, 8 μL of PNGase F enzyme is added to the sample, which is subsequently incubated at 37 °C for 5 h for desugarization. After incubation, the sample is centrifuged at 12000 rpm for 5 min, and the resulting supernatant is added to a sample vial as a test sample for subsequent testing. Chromatography column: PolyLC™ PolyHYDROXYETHYLA column, 300 Å, 5 μm, 2.1 mm×200 mm; Mobile phase: 50 mM ammonium acetate, pH 7.0; Running time: 10 minutes; Flow rate: 0.1mL / min; Loading volume: 2 μL; Column temperature: 25°C; Detection wavelength: 280nm; Ionization mode: ESI positive; Drying gas temperature: 325℃; Drying gas flow rate: 8L / min; Atomizer pressure: 20psig; Sheath gas temperature: 325℃; Sheath gas flow rate: 12L / min; Scan settings: 900 m / z~8000 m / z.

[0343] d. Size Exclusion Chromatography for Determining Molecular Size Heterogeneity (SEC-HPLC) Sample processing: The sample is diluted to 1.0 mg / mL with the mobile phase, then centrifuged at 12000 rpm for 10 minutes, and the resulting supernatant is subjected to analysis. Chromatography column: Tosoh Corporation, TSKgel G3000SWXL, 5 μm, 7.8 mm × 300 mm; Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8); Flow rate: 0.6mL / min; Detection wavelength: 280nm; Column temperature: 30°C; Loading volume: 20 μL; Elution time: 20 min; Elution gradient: isocratic elution.

[0344] e. Non-reduced capillary electrophoresis-sodium dodecyl sulfate (NR-CE-SDS) to determine purity The determination is carried out according to the method described in the Chinese Pharmacopoeia, Part IV General Provisions 3127, "Determination of molecular size variants of monoclonal antibodies."

[0345] f. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) for Determining Drug-to-Antibody Ratios (DAR Values) of Antibody-Drug Conjugates The analysis is carried out according to the method described in US Pat. No. 10,227,417.

[0346] Formula for calculating DAR: DAR=2×(Σ(light chain weighted peak area)+Σ(heavy chain weighted peak area)) / 100, i.e., DAR=2×(L0 peak area ratio×0+L1 peak area ratio×1+H0 peak area ratio×0+H1 peak area ratio×1+H2 peak area ratio×2+H3 peak area ratio×3) / 100.

[0347] g. Reverse-phase high performance liquid chromatography (RP-HPLC) for determining the drug-antibody ratio (DAR value) of antibody-drug conjugates Sample pretreatment: Samples are treated according to the method described in US Pat. No. 10,227,417. HPLC equipment: Waters Acquity Arc; Mobile phase A: 0.1%TFA+ACN; Mobile phase B: 0.1%TFA+H2O; Analytical column: Waters; BioResolve; 2.1 × 100 mm; 2.7 μm; 450 Å; PN 0024168; Loading volume: 5 μL; Flow rate: 0.25mL / min; Column temperature: 80°C; Detector: PDA detector; Detection wavelength: 280nm. Chromatographic gradient used:

[0348] [Table 6]

[0349] Example 4.1 Preparation and characterization of antibody-drug conjugates targeting Trop-2 The anti-Trop-2 antibodies hTINA1 (made with reference to the sequence of datopotamab provided in the WHO Drug Information) and h23-12 (100 mg each) were conjugated with drug-containing linkers MWD-L1, MWC-L2, MWC-L3, MWF-L6, MWD-L7, MWD-L8, MWD-L9 and MWF-L8, respectively, according to the method described in Group 4 of this Example to obtain crosslinked ADCs 1a, 1b, 1c, 1d, 1j, 1k, 1l, 1m, 1n, 1o and 1p. An intermediate sample obtained during the preparation of ADC 1d that was not subjected to hydrophobic chromatography was kept and named intermediate sample 1j.

[0350] The anti-Trop-2 antibodies hTINA1 and h23-12 (100 mg each) were conjugated with drug-containing linkers MC-GGFG-Dxd, LA, LB, LJ, MWS-L7, LI and MWS-L6, respectively, according to the method described in China Patent No. 105849126 to obtain stochastically conjugated ADCs 1e, 1f, 1g, 1h, 1i, 1q, 1r, 1s, 1t and 1u.

[0351] The DAR values, concentrations, and purity of the crosslinked ADCs (1a-1d, 1j, and 1k-1p) were determined using UV spectrophotometry as described in Section 4.2a, hydrophobic chromatography as described in Section 4.2b, and size exclusion chromatography as described in Section 4.2d of Group 4 of this Example.

[0352] The DAR values ​​of ADCs conjugated with the drug-containing linker MC-GGFG-Dxd (1e, 1h, 1q, 1r, 1s, 1t and 1u) were determined using RP-HPLC as described in Section 4.2f of Group 4 of this Example, the DAR values ​​of ADCs conjugated with the drug-containing linkers LA or LB (1f, 1g and 1i) were determined using RP-HPLC as described in Section 4.2g of Group 4 of this Example, and the concentration and purity of the uncrosslinked ADCs (1c-1i) were determined using UV spectrophotometry as described in Section 4.2a and size exclusion chromatography as described in Section 4.2d of Group 4 of this Example.

[0353] The results are presented in Table 1.

[0354] [Table 7]

[0355] (In this disclosure, h23-12 is a monoclonal antibody having the following sequence: Heavy chain variable region: QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG EITPSDNYGSYNQKFKG RVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDY WGQGTLVTVSS (SEQ ID NO: 7) Heavy chain CDR1: SYWMH (SEQ ID NO: 1) Heavy chain CDR2: EITPSDNYGSYNQKFKG (SEQ ID NO: 2) Heavy chain CDR3: GHGNYVSFDY (SEQ ID NO:3) Light chain variable region: DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLIY YTSRLES GVPSRFSGSGSGTDFLTISSLQPEDFATYFC QQGYTLPPYT FGQGTKLEIK (SEQ ID NO: 8) Light chain CDR1: RASQDISNYLN (SEQ ID NO: 4) Light chain CDR2: YTSRLES (SEQ ID NO:5) Light chain CDR3: QQGYTLPPYT (SEQ ID NO: 6) Heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) Light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10)

[0356] Example 4.2 Preparation of HER-2 Targeted Antibody-Drug Conjugates The anti-HER-2 antibody trastuzumab (CAS:180288-69-1, purchased from Shanghai Roche Pharmaceutical Co., Ltd.) (100 mg) was conjugated to each of the drug-containing linkers MWD-L1, MWD-L2, MWE-L4, MWF-L6, MWD-L8, MWD-L9, LC, and MWC-L3 according to the methods described in Group 4 of this Example to give the crosslinked ADCs 2a, 2b, 2c, 2h, 2i, 2d, and 2l.

[0357] The anti-HER-2 antibody trastuzumab (100 mg) was conjugated with the drug-containing linkers MC-GGFG-DXD, LA and LB, respectively, according to the method described in Chinese Patent No. 105829346 to obtain the stochastically conjugated ADCs 2e, 2f, 2g, 2k and 2m.

[0358] The DAR values, concentrations and purity of the crosslinked ADCs (2a-2d, 2h-2j and 2l) were determined using UV spectrophotometry as described in Section 4.2a, hydrophobic chromatography as described in Section 4.2b and size exclusion chromatography as described in Section 4.2d of Group 4 of this Example.

[0359] The DAR values ​​of the ADCs conjugated with the drug-containing linker MC-GGFG-Dxd were determined using RP-HPLC as described in Section 4.2f of Group 4 of this Example, the DAR values ​​of the ADCs conjugated with the drug-containing linkers LA or LB (2e and 2b) were determined using RP-HPLC as described in Section 4.2g of Group 4 of this Example, and the DAR values ​​and the concentration and purity of the non-crosslinked ADCs (2e-2g and 2k and 2m) were determined using UV spectrophotometry as described in Section 4.2a and size exclusion chromatography as described in Section 4.2d of Group 4 of this Example.

[0360] The results are presented in Table 2.

[0361] [Table 8]

[0362] Example 4.3 Structural characterization of compounds The Trop-2-targeting ADCs (1a, 1h, and 1j) prepared in Example 4.1 and the HER-2-targeting ADCs (2b and 2e) prepared in Example 4.2 were subjected to analysis by hydrophobic chromatography as described in Section 4.2b of Group 4 of this Example to compare the heterogeneity of the ADCs prepared by the different conjugation methods.

[0363] The ADCs are shown in Table 3.

[0364] [Table 9]

[0365] Comparison of Trop2-ADC-4', Trop2-ADC-4 and Trop2-ADC-8 shows that Trop2-ADC-4 and Trop2-ADC-4' prepared with the drug-containing linker MWD-L1 had better drug uniformity compared to the existing ADC (Trop2-ADC-8) prepared with the drug-containing linker MC-GGFG-Dxd. The results are presented in Figure 1.

[0366] Comparison of Her2-ADC-2 and Her2-ADC-5 shows that Her2-ADC-2, an ADC prepared using the drug-containing linker MWC-L2, had better drug uniformity than Her2-ADC-5, which was prepared using the existing drug-containing linker LA.

[0367] Example Group 5 Activity Evaluation Example 5.1 Comparative Study of Camptothecin Activity KB oral epithelial carcinoma cells (purchased from ATCC) were cultured in DMEM medium supplemented with 10% FBS, NCI-N87 human gastric carcinoma cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS, HT29 human colon carcinoma cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS, and BxPC-3 human pancreatic adenocarcinoma cells (purchased from ATCC) were cultured in RPMI1640 medium supplemented with 10% FBS.

[0368] Using the corresponding medium, KB cells were grown in cell plates at a density of 2 × 10 4 The density of NCI-N87 cells was adjusted to 5 × 10 4 The density of HT29 cells was adjusted to 5 × 10 cells / mL. 4 The cells were then plated in a 96-well clear flat-bottom plate at 100 μL / well and cultured overnight to a density of 5 × 10 BxPC-3 cells. 4 The concentration was adjusted to 1 / mL.

[0369] The samples to be detected were diluted to 20 μM with the corresponding medium, and then 5-fold gradient dilutions were performed to obtain 9 concentrations. The wells with cells but without any sample were set as wells showing maximum cell proliferation, and the wells without cells were set as wells with medium background. The diluted samples were added to the cell plate at 100 μL / well and then incubated in a CO2 incubator for 96 hours. After incubation, pre-prepared CCK-8 (purchased from Dojindo Laboratories, Inc.) was added to the plate at 20 μL / well, and after further incubation in a CO2 incubator for 3 hours, the OD values ​​were read by a microplate reader.

[0370] The data obtained were fitted to a four-parameter curve using the software Prism7, and the formula for calculating maximum killing was: Maximum killing (%) = 1 - (OD450 value of the well showing maximum killing - OD450 value of the well with medium background) / (OD450 value of the well showing maximum cell growth - OD450 value of the cells with medium background) x 100%. The results are shown in Table 4.

[0371] [Table 10-1] [Table 10-2]

[0372] A comparative study was performed on antibody-drug conjugates conjugated with different drug-containing linkers and is presented below.

[0373] Example 5.2 Comparative study of the activity of HER-2-targeting ADCs conjugated with different drug-containing linkers N87 cell line (purchased from ATCC) with high expression of HER-2 was removed from liquid nitrogen and harvested. The cell density was then adjusted to 5 × 10 in complete medium. 4 The concentration was adjusted to cells / mL, and the cells were added to a cell culture plate (a 96-well plate was used, and sterile PBS or sterile purified water was added to rows A and H and columns 1 and 12 of the plate at 200 μL / well) at 100 μL / well and cultured overnight.

[0374] The samples of ADC targeting HER-2 prepared above were diluted to 50 μg / ml each using complete medium, followed by 4-fold gradient dilution to obtain 9 concentrations and zero concentration. The diluted samples were added to a cell culture plate, and three duplicate wells were set up for each of the diluted samples, and negative control wells (cells + medium) and blank control wells (no cells, only medium) were set up in 11 columns of the plate. Then, the plate was placed in a cell incubator, and the cells were incubated for 120 hours or 168 hours (see the table below for details). After incubation, MTS was added to the plate at 40 μL / well, and then it was returned to the incubator at 37 °C and reacted for 2 hours to 4 hours. The cell plate was then taken out, and the OD value at 490 nm was read with a microplate reader.

[0375] The results are shown in Table 5.

[0376] [Table 11]

[0377] The above results indicate that the efficacy of the ADC prepared using the drug-containing linker MWD-L1 was stronger than that of the ADC prepared using the existing drug-containing linkers MC-GGFG-Dxd and LA, and the efficacy of the ADC prepared using the self-made LC for cross-linking conjugation was inferior to that of the ADC prepared using the drug-containing linkers MWD-L1 and MC-GGFG-Dxd.

[0378] In addition, further comparisons were made between ADCs prepared using the drug-containing linker MWC-L2 and those prepared using LB and MC-GGFG-Dxd, as described above, and the results are shown in Table 6.

[0379] [Table 12]

[0380] The above results indicate that the drug-containing linker MWC-L2 also has certain advantages in cell killing in vitro compared with the existing drug-containing linkers LB and MC-GGFG-Dxd.

[0381] In addition, as described above, further comparisons were made between the drug-containing linker MWD-L1 and the drug-containing linkers MWD-L8 and MWD-L9. The results are shown in Table 7.

[0382] [Table 13]

[0383] The above results indicate that the efficacy of the ADCs prepared with the drug-containing linker MWD-L1 (phenylene substituted) was more pronounced compared to that of the ADCs prepared with the drug-containing linkers MWD-L8 (phenylene unsubstituted) and MWD-L9 (phenylene substituted with trifluoromethyl).

[0384] Example 5.3 Comparative study of the activity of Trop-2-targeting ADCs conjugated with different drug-containing linkers BxPC3 cell line (purchased from ATCC) with high expression of Trop-2 was removed from liquid nitrogen and harvested. The cell density was then adjusted to 5 × 10 4 The concentration was adjusted to cells / mL, and the cells were added to a cell culture plate (a 96-well plate was used, and sterile PBS or sterile purified water was added to rows A and H and columns 1 and 12 of the plate at 200 μL / well) at 100 μL / well and cultured overnight.

[0385] The samples of ADCs targeting Trop-2 prepared above were diluted to 50 μg / ml each using complete medium, followed by 4-fold gradient dilution to obtain 9 concentrations and zero concentration. The diluted samples were added to a cell culture plate, and three duplicate wells were set up for each of the diluted samples, and negative control wells (cells + medium) and blank control wells (no cells, only medium) were set up in 11 columns of the plate. Then, the plate was placed in a cell incubator, and the cells were incubated for 120 hours. After incubation, MTS was added to the plate at 40 μL / well, and then it was returned to the incubator at 37 °C and reacted for 2 to 4 hours. The cell plate was then taken out, and the OD value at 490 nm was read with a microplate reader.

[0386] The results are shown in Table 8.

[0387] [Table 14]

[0388] The above results show that the ADC prepared using the drug-containing linker MWD-L1 exhibited efficacy equivalent to that of the ADC prepared using MWC-L2, and that the efficacy of the ADC prepared using the drug-containing linker according to the present invention was superior to that of the ADC prepared using the existing linkers LB, LA, and MC-GGFG-Dxd.

[0389] In addition, further comparisons were made between Trop2-ADC-1, Trop2-ADC-10 and Trop2-ADC-6 as described above, and the results are shown in Table 9.

[0390] [Table 15]

[0391] The results of the above comparative study show that there were significant differences in the activity of ADCs prepared using drug-containing linkers with different drug release structures, and the ADC using MWD-L1 had the best efficacy.

[0392] In addition, further comparisons were made between Trop2-ADC-1, Trop2-ADC-9 and Trop2-ADC-8 as described above, and the results are shown in Table 10.

[0393] [Table 16]

[0394] The above results indicate that the novel ADC Trop2-ADC-1 provided by the present invention has certain advantages in activity compared to Trop2-ADC-8 and Trop2-ADC-9.

[0395] Example 5.4 In vitro efficacy study of different ADCs against tumors expressing low abundance antigens and against cells with low antigen expression in the vicinity of tumor cells Due to the heterogeneity of progressive tumors, tumor tissues have variable antigen expression.

[0396] The bystander effect of tumors expressing low abundance antigens and cells with low antigen expression near tumor cells usually affects the prognosis of patients with advanced tumors and is an important indicator for drug evaluation. Therefore, tumors expressing low abundance antigens and cells with low antigen expression near tumor cells were evaluated, respectively.

[0397] (1) In vitro efficacy study of different ADCs against tumors expressing low-abundance antigens HS-746T cells were purchased from ATCC. The cells were cultured in RPMI1640 / IMEM (1:1) containing 10% fetal bovine serum (FBS) supplemented with penicillin and streptomycin, and cultured in an incubator at 37°C in air containing 5% CO2.

[0398] After seeding, the cells were treated with 0.1 μg / mL and 1 μg / mL Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-5, Trop2-ADC-10 and Trop2-ADC-14 for 168 hours, respectively, and then harvested and counted. Next, the cells were incubated with the above-mentioned Trop-2-targeting Dylight 488 NHS Ester-labeled ADC in the dark at 4 °C for 1 hour, centrifuged to remove the supernatant, resuspended in phosphate buffer (PBS, pH 7.4), washed three times with PBS (pH 7.4), and the number of HS-746T cells was detected and calculated using a flow cytometer BD ACCURI C6 PLUS.

[0399] [Table 17]

[0400] From the above study, it can be seen that the ADCs prepared using the drug-containing linkers MWD-L1, MWC-L2 and MWF-L6 all showed a certain killing effect on HS-746T tumor cells expressing low-abundance antigens. The ADCs prepared by the two conjugation methods using the drug-containing linkers GGFG-DXD and MWD-L7 showed little killing effect on tumors expressing such low-abundance antigens. In addition, the drug-containing linker containing compound 3 had a better killing effect than the small molecule MWC-1. This may be related to the better cell membrane permeability of compound 3 itself.

[0401] (2) In vitro efficacy study of different ADCs against cells with low antigen expression near tumor cells KPL-4 and MDA-MB-468 cells were purchased from ATCC. The cells were cultured in RPMI1640 / IMEM (1:1) containing 10% fetal bovine serum (FBS) supplemented with penicillin and streptomycin, and incubated in an incubator at 37°C in air containing 5% CO2.

[0402] HER2-positive KPL-4 cells and HER2-negative MDA-MB-468 cells were seeded together or HER2-negative MDA-MB-468 cells were seeded separately, and then all cells were treated with ADC for 168 hours. Then, the cells were harvested and incubated with Dylight 488 NHS Ester-labeled anti-HER2 antibody in the dark at 4°C for 1 hour. Next, the cells were centrifuged to remove the supernatant, resuspended in PBS (pH 7.4), washed three times with PBS, and the cell ratios of KPL-4 cells and MDA-MB-468 cells were detected and calculated using a flow cytometer BD ACCURI C6 PLUS, and the number of the two types of cells was calculated.

[0403] [Table 18]

[0404] From the above study, it can be seen that all the groups treated with high concentration ADC (1 μg / mL) showed tumor inhibition effect on antigen-negative cells. However, comparison of antigen-positive and -negative cells in the groups treated with different concentrations of ADC showed that the bystander killing effect of MWF-L6 and MWC-L2 was better than that of the control GGFG-DXD and LB.

[0405] Example 5.5 Study of the in vivo killing effect of different drug-containing linkers BxPc-3 human pancreatic cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences were used. BxPC-3 cells were cultured in RPMI1640 containing 10% fetal bovine serum supplemented with penicillin and streptomycin at 37°C in a 10 cm culture dish for adherent culture in an incubator containing 5% CO2. Cells were passaged 2-3 times a week, and when they reached the exponential growth phase, they were digested with trypsin, harvested, counted, and seeded.

[0406] A comparative study was conducted between different dose groups of antibody-drug conjugates Trop2-ADC-1, Trop2-ADC-2, Trop2-ADC-3 and Trop2-ADC-5 prepared with different drug-containing linkers (Table 13). Mice were administered these ADCs by intravenous injection (IV) at a dose volume of 10 mL / kg, and mice in the vehicle group were administered the same volume of vehicle (saline). The specific doses and administration schedules are shown in Table 13. Tumor volumes were measured twice a week, mice were weighed, and data were recorded.

[0407] The experiment is completed when the experiment reaches its endpoint or the tumor volume reaches 2000 mm 3 When the tumor reached 100%, the animals were sacrificed under CO2 anesthesia and the tumors were dissected and photographed. The results are shown in Table 13 and Figure 2.

[0408] [Table 19-1] [Table 19-2]

[0409] The above results indicate that the in vivo activities of all the drug-containing linkers MWD-L1, MWC-L2 and MWC-L3 for site-specific conjugation were superior to that of the control molecule GGFG-DXD. Meanwhile, MWC-L2 and MWC-L3, which have the dipeptide structure of VA and the tetrapeptide releasing structure of GGFG, respectively, had similar levels of efficacy in vivo.

[0410] Example 5.6 In vivo efficacy studies of antibody-drug conjugates targeting Trop-2 HT1376 human bladder cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences were used. HT1376 cells were cultured in RPMI1640 containing 10% fetal bovine serum supplemented with penicillin and streptomycin at 37°C in a 10 cm culture dish for adherent culture in an incubator containing 5% CO2. Cells were passaged 2-3 times a week, and when they reached the exponential growth phase, they were digested with trypsin, harvested, counted, and seeded.

[0411] A comparative study was conducted between groups with different doses of an exemplary antibody-drug conjugate, Trop2-ADC-14 (Table 14). Mice were administered the ADC via intravenous injection (IV) at a dose volume of 10 mL / kg, and mice in the vehicle group were administered the same volume of vehicle (saline). The specific doses and administration schedules are shown in Table 14. Tumor volumes were measured twice a week, mice were weighed, and the data were recorded.

[0412] The experiment is completed when the experiment reaches its endpoint or the tumor volume reaches 2000 mm 3 When the tumor volume reached 100 mg / kg, the animals were sacrificed under CO2 anesthesia, and the tumors were dissected and photographed. The results are shown in Table 14 and Figure 3, which shows the growth changes of the tumor volume in each group of mice.

[0413] [Table 20]

[0414] The above results indicate that in the xenograft tumor model of HT1376 human bladder cancer, Trop2-ADC-14 exhibited obvious tumor-inhibitory activity compared with the vehicle group, while the tumor-inhibitory activity of Trop2-ADC-14 was obviously superior to that of DS-1062 and Trodelvy™ at the same dose.

[0415] Example 5.7 In vivo efficacy studies of antibody-drug conjugates targeting Trop-2 Calu-3 lung cancer cells purchased from the Cell Bank of the Chinese Academy of Sciences were used. Calu-3 cells were cultured in RPMI1640 containing 10% fetal bovine serum supplemented with penicillin and streptomycin at 37°C in a 10 cm culture dish for adherent culture in an incubator containing 5% CO2. The cells were passaged 2-3 times a week, and when they reached the exponential growth phase, they were digested with trypsin, harvested, counted, and seeded.

[0416] A comparative study was conducted between groups with different doses of an exemplary antibody-drug conjugate, Trop2-ADC-14 (Table 15). Mice were administered the ADC by intravenous injection (IV) at a dose volume of 10 mL / kg, and mice in the vehicle group were administered the same volume of vehicle (saline). The specific doses and administration schedules are shown in Table 15. Tumor volumes were measured twice a week, mice were weighed, and the data were recorded.

[0417] The experiment is completed when the experiment reaches its endpoint or the tumor volume reaches 2000 mm 3 When the tumor volume reached 100 mg / kg, the animals were sacrificed under CO2 anesthesia, and the tumors were dissected and photographed. The results are shown in Table 15 and Figure 4, which shows the growth changes of the tumor volume in each group of mice.

[0418] [Table 21]

[0419] The above results indicate that Trop2-ADC-14 exhibited obvious tumor-inhibitory activity in the Calu-3 human lung cancer xenograft tumor model compared with the vehicle group, while the tumor-inhibitory activity of Trop2-ADC-14 was superior to that of DS-1062 and Trodelvy™ at the same dose.

[0420] Example 5.8 Bystander Effect Study of Antibody-Drug Conjugates Targeting Trop-2 In a 6-well plate, Trop-2 positive BxPC-3 cells (purchased from ATCC) and Trop-2 negative HT-29 cells (purchased from ATCC) were seeded together or Trop-2 negative HT-29 cells were seeded separately. After treatment with Trop2-ADC-14 (10ng / mL, 30ng / mL and 100ng / mL) or DS-1062a (100ng / mL and 300ng / mL) for 144 hours, the cells were harvested and counted. The cells were then incubated with Dylight 488 NHS Ester-labeled Trop2-ADC-14 on ice in the dark for 1 hour, centrifuged to remove the supernatant, resuspended in PBS, washed three times with PBS, and the cell ratios of BxPC-3 cells and HT-29 cells were detected and calculated using a flow cytometer BD ACCURI C6 PLUS, and the number of the two types of cells was calculated. The results are shown in Figure 5.

[0421] The results showed that Trop2-ADC-14 had a strong bystander effect, killing Trop-2-negative cells while killing Trop-2-positive cells, and the bystander killing effect of Trop2-ADC-14 at 30 ng / mL was comparable to that of the reference drug DS-1062a at 300 ng / mL (Figure 5A), suggesting that the bystander effect of Trop2-ADC-14 was overall stronger than that of the reference drug DS-1062a. Neither Trop2-ADC-14 nor DS-1062a had any obvious effect on the proliferation of Trop-2-negative HT-29 cells cultured separately (Figure 5B).

[0422] Example 5.9 Study of the growth inhibitory effects of Trop-2-targeting antibody-drug conjugates and camptothecin on different tumor cells For cells growing adherently, the SRB assay was used to detect the effect of antibody-drug conjugates or camptothecin on the proliferation of tumor cells cultured in vitro. A certain number of cells in the exponential growth phase were seeded in a 96-well cell culture plate, and different concentrations of antibody-drug conjugates or camptothecin were added to the cells when the cells were grown adherently overnight. After 144 hours, the cells were fixed with trichloroacetic acid and then stained with SRB (prepared at a concentration of 4 mg / mL in 1% glacial acetic acid). 10 mM Tris solution was added to each well to solubilize the bound SRB. The OD value at 510 nm was read on a microplate reader. Inhibition (%) = (OD value of control wells - OD value of treated wells) / OD value of control wells x 100%

[0423] For cells growing in suspension, MTT assay was used to detect the effect of antibody-drug conjugates or camptothecin on the proliferation of tumor cells cultured in vitro. A certain number of cells in exponential growth phase were seeded in 96-well cell culture plates, and different concentrations of antibody-drug conjugates or camptothecin were added to the cells when the cells were grown in suspension overnight. After 144 hours, MTT was added to each well of the plate, and incubation was continued for 4 hours at 37°C in a 5% CO2 saturated humidity incubator. Then, 100 μL of SDS-isobutanol-HCl solution was added to each well of the plate, and the OD values ​​at 570 nm and 690 nm were read with a microplate reader.

[0424] The 50% inhibitory concentration (IC50) was calculated from the inhibition rate (%) at these different concentrations using the software Graphpad Prism 8.0. The experiment was repeated once and the data was expressed as the mean ± SD. The results are shown in Table 16.

[0425] [Table 22]

[0426] Example 5.10 Study of the internalization function of antibody-drug conjugates targeting Trop-2 BxPC-3 cells (purchased from ATCC) were seeded in 6-well plates and 1 μg / mL of fluorescently labeled Trop2-ADC-14 and mAb in Trop2-ADC-14 (i.e. antibody h23-12) were added. The cells were incubated at 37°C for 3, 6, 12, 18, 24 and 48 hours, respectively, and then digested with trypsin. Uninternalized Trop2-ADC-14 and mAb h23-12 were washed away, and the fluorescence intensity was measured with a flow cytometer (BD ACCURI C6 PLUS). The experiment was repeated once. The results are shown in Figure 6.

[0427] Studies have shown that when incubated with Trop-2 positive BxPC-3 cells, fluorescently labeled Trop2-ADC-14 and h23-12 were internalized into the cells. Internalization was time-dependent, with the amount of internalized drug increasing over time, and Trop2-ADC-14 was internalized to a greater extent than the antibody h23-12 at 24 hours.

[0428] Example 5.11 Study of the apoptosis-inducing effects of antibody-drug conjugates targeting Trop-2 and camptothecin BxPC-3 cells (purchased from ATCC) were seeded in 6-well plates and then treated with Trop2-ADC-14 (0.020nM, 0.196nM and 1.963nM), DS-1062 (0.020nM, 0.196nM and 1.963nM), mAb (i.e. antibody h23-12) in Trop2-ADC-14 (6.728nM) and compound 3 (0.1nM, 1nM and 10nM) for 120 hours. Then, the cells were harvested, Annexin-V-FITC and PI were added thereto, and the cells were stained for 15 minutes at room temperature in the dark, and finally, resuspended in 300μl of 1× binding buffer added. Apoptosis was detected by a flow cytometer (BD Accuri™ C6 Plus flow cytometer) and 1×10 4Cells were gated for each group. Experimental data were analyzed using the software BD CSampler™ Plus C6. The results are shown in Figure 7.

[0429] The study shows that Trop2-ADC-14, even at 0.196 nM, clearly induced the degradation of the apoptosis marker protein pro-PARP and induced the hydrolysis of procaspase 3 to the active protein caspase 3 (cleaved caspase 3), and its apoptosis-inducing effect was concentration-dependent. The apoptosis-inducing effect of Trop2-ADC-14 was obviously stronger than that of DS-1062a at the same concentration, which showed no obvious apoptosis-inducing effect at 0.196 nM. The small molecule compound, namely compound 3, similarly induced apoptosis of BxPC-3 cells in a concentration-dependent manner, and the antibody h23-12 showed no significant apoptosis-inducing effect on BxPC-3 cells.

[0430] Example 5.12 Study of the binding activity of Trop-2-targeting antibody-drug conjugates to Trop-2-positive cells Different concentrations (1ng / mL, 3ng / mL, 10ng / mL, 30ng / mL, 100ng / mL, 300ng / mL, 1000ng / mL, 3000ng / mL, 10000ng / mL, 30000ng / mL and 100000ng / mL) of fluorescently labeled Trop2-ADC-14 and mAb in Trop2-ADC-14 (i.e. antibody h23-12) were incubated with BxPC-3 cells (purchased from ATCC) in the dark for 1 hour on ice, after which the cells were centrifuged to remove the supernatant, washed and resuspended in PBS, and the fluorescence intensity of the drug bound to the cells was measured by flow cytometer (BD ACCURI C6 PLUS). The experiment was repeated once. The results are shown in Table 17 and Figure 8.

[0431] [Table 23]

[0432] The study showed that both Trop2-ADC-14 and antibody h23-12 showed concentration-dependent binding to Trop-2 positive BxPC-3 tumor cells with EC50 values ​​of 1296.0±155.6ng / mL and 1137.5±128.0ng / mL, respectively, indicating comparable levels of binding ability to Trop-2 positive cells. In contrast, neither Trop2-ADC-14 nor antibody h23-12 had any obvious binding to Trop-2 negative HT-29 cells, indicating that the binding of Trop2-ADC-14 and antibody h23-12 to tumor cells was dependent on the expression level of Trop-2.

[0433] The above description of the embodiments of the present disclosure is not intended to limit the present disclosure, and those skilled in the art may make various changes and modifications to the present disclosure without departing from the spirit of the present disclosure, which should be included in the scope of the appended claims.

Claims

1. Structural formula I: 【Chemistry 1】 (In structural formula I, R １ , R ２ , R ３ and R ４ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or R １ , R ２ , R ３ and R ４ any two of these together with the carbon atoms to which they are attached form a C3-6 cycloalkyl; G is hydrogen, halogen, methyl or methoxy; Y is oxygen, sulfur, sulfone, sulfoxide, methylene, or substituted methylene; X is oxygen or sulfur; wherein n=0 or 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

2. The compound has structural formula IA: 【Chemistry 2】 (In structural formula IA, R １ , R ２ , R ３ and R ４ and n is not simultaneously hydrogen), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

3. Structural formula II: 【Transformation 3】 (In structural formula II, R ５ is C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cycloalkyl or C3-6 cycloalkyl substituted with one or more substituents, phenyl or substituted phenyl; G is hydrogen, halogen, methyl or methoxy; X is oxygen or sulfur; n=0 or 1; When X is oxygen, G is hydrogen, and n=0, R ５ is not n-butyl), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

4. The compound has structural formula IIA: 【Chemistry 4】 (In structural formula IIA, R ５ The compound according to claim 3, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that it is a compound represented by the formula (I) wherein R is not n-butyl.

5. The compound has the following structure: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 5. The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that it has one of the following formula (1):

6. Structural formula III: 【Transformation 6】 wherein E is 【Transformation 7】 means the bonding site to M, 【Transformation 8】 is selected from the group consisting of M is phenylene or phenylene substituted with one or more substituents, or a chemical bond, wherein in the substituted phenylene, the phenylene is substituted with substituent(s) selected from the group consisting of alkyl, haloalkyl, alkoxy, halogen, ester, amide, and cyano, preferably M is halogen-substituted phenylene; SP １ is selected from the group consisting of C1-8 alkylene, C1-8 cycloalkylene, or C1-21 straight-chain heteroalkylene containing 1 to 11 heteroatoms selected from the group consisting of N, O, and S, wherein the C1-8 alkylene, C1-8 cycloalkylene, and C1-21 straight-chain heteroalkylene are optionally substituted with one or more substituents independently selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano; SP ２ is -NH(CH ２ CH ２ O) ａ CH ２ CH ２ CO-, -NH(CH ２ CH ２ O) ａ CH ２ CO-, -S(CH ２ ) ａ CO- or a chemical bond, where a is an integer ranging from 1 to 20, preferably an integer ranging from 1 to 10, more preferably an integer ranging from 1 to 6; A represents a peptide group of 2 to 4 amino acids, CPT is a compound of camptothecin) or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

7. The compound has structural formula IIIA: 【Chemistry 9】 (In Structural Formula IIIA, R ６ and R ７ are each independently hydrogen, halogen, or Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents, wherein the phenyl is substituted with a substituent(s) selected from the group consisting of alkyl, alkoxy, halogen, ester, amide, and cyano; Preferably, Ar' is phenyl, phenyl substituted with 4-formylmethylamine ( 【Chemistry 10】 ) or phenyl substituted with 4-formylmorpholine ( 【Chemistry 11】 7. The compound of claim 6, wherein R is a methyl group, R is a methyl group, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

8. In Formula III or Formula IIIA, CPT is represented by Formula I: 【Chemistry 12】 (In structural formula I, the groups G, X, Y, R １ , R ２ , R ３ , R ４ and n is a group G, X, Y, R １ , R ２ , R ３ , R ４ and n has the same meaning as defined in claim 1), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, Preferably, the CPT has structural formula IA: 【Chemistry 13】 (In structural formula IA, the group R １ , R ２ , R ３ and R ４ is a group R １ , R ２ , R ３ and R ４ has the same meaning as defined in claim 2, except that R １ , R ２ , R ３ and R ４ and R may simultaneously be hydrogen), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, 8. The compound of claim 6 or 7, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, wherein the compound represented by formula I or formula IA is bonded to the carboxyl of A through an amide bond via its amino in formula III or formula IIIA.

9. In Formula III or Formula IIIA, CPT is represented by Formula II: 【Chemistry 14】 (In structural formula II, the groups G, R ５ , X and n are groups G, R ５ , X and n have the same meanings as defined in claim 3), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, Preferably, the CPT has structural formula IIA: 【Chemistry 15】 (In structural formula IIA, the group R ５ is a group R ５ has the same meaning as defined in claim 4, except that the group R ５ or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof according to claim 6 or 7, characterized in that it is a compound represented by the formula (I), wherein R is an integer of 1 to 3, and R is an integer of 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

10. In Formula III or Formula IIIA, CPT is represented by Formula IV: 【Chemistry 16】 (In structural formula IV, R ８ is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cycloalkyl or C3-6 cycloalkyl substituted with one or more substituents, or halogen), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof; In the formula III or IIIA, the compound represented by the formula IV is ８ 8. The compound of claim 6 or 7, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that it is bonded to the carboxyl of A by a self-releasing structure via its hydroxyl bonded to the same carbon as

11. The compound represented by Formula III or Formula IIIA may further comprise a compound represented by Formula V: 【Chemistry 17】 (In structural formula V, R ６ and R ７ are each independently Ar'S, where Ar' is phenyl or phenyl substituted with one or more substituents; Xh and Yh are each independently hydrogen, halogen, haloalkyl, or alkoxy, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.

12. The compound represented by structural formula V may further be represented by structural formula VA: [Chemistry 18] is a compound represented by Preferably, the compound represented by the structural formula VA further comprises the structural formula VA-1: 【Chemistry 19】 is a compound represented by Alternatively, the compound represented by formula V may further comprise a compound represented by formula VB: 【Chemistry 20】 is a compound represented by Preferably, the compound represented by the structural formula VB further comprises the compound represented by the structural formula VB-1: 【Chemistry 21】 is a compound represented by Alternatively, the compound represented by formula V may further be represented by formula VC: 【Chemistry 22】 12. The compound according to claim 11, characterized in that it is a compound represented by: or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

13. The compound has structural formula VI: 【Chemistry 23】 is a compound represented by Preferably, the compound represented by structural formula VI further comprises structural formula VI-A: 【Chemistry 24】 is a compound represented by Preferably, the compound represented by structural formula VI-A further comprises structural formula VI-A-1: 【Chemistry 25】 is a compound represented by Alternatively, the compound represented by structural formula VI may further comprise structural formula VI-B: 【Chemistry 26】 is a compound represented by Preferably, the compound represented by structural formula VI-B further comprises a compound represented by structural formula VI-B-1: 【Chemistry 27】 is a compound represented by Alternatively, the compound represented by structural formula VI may further be represented by structural formula VI-C: 【Chemistry 28】 The compound according to any one of claims 6 to 12, characterized in that it is a compound represented by: or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

14. The compound has the following structure: 【Chemistry 29-1】 【Chemistry 29-2】 【Chemistry 29-3】 【Chemistry 29-4】 【Chemistry 29-5】 【Chemistry 29-6】 【Chemistry 29-7】 【Chemistry 29-8】 【Chemistry 29-9】 [Chemistry 29-10] 14. The compound according to any one of claims 6 to 13, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that it has one of the following:

15. An antibody-drug conjugate prepared using the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof and an antibody or a fragment thereof, Preferably, the antibody-drug conjugate has the general formula: 【Transformation 30】 (wherein mAb means an antibody or a fragment thereof; Ki M, SP １ , SP ２ , A and CPT are groups M, SP １ , SP ２ , A and CPT have the same meanings as defined in any one of claims 6 to 14, and N ranges from 1 to 10, preferably from 1 to 8, more preferably from 3 to 8; E Ｌ teeth, 【Chemistry 31】 means a bond with a cysteine ​​in the mAb, 【Chemistry 32】 means a bond to M, 【Transformation 33】 An antibody-drug conjugate having a structure represented by the formula:

16. General formula VII or general formula VIII as follows: 【Transformation 34】 (In general formula VII and general formula VIII, N is in the range of 1 to 10, preferably 1 to 8, more preferably 3 to 8).

17. The antibody-drug conjugate of claim 15 or 16, wherein the antibody or fragment thereof is an anti-Trop-2 antibody or fragment thereof, or an anti-HER-2 antibody or fragment thereof; Preferably, the anti-Trop-2 antibody or fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and light chain CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; more preferably, the anti-Trop-2 antibody or fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8; even more preferably, the anti-Trop-2 antibody or fragment thereof further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:9 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO:10; particularly preferably, the anti-Trop-2 antibody or fragment thereof is a monoclonal antibody or fragment thereof; Alternatively, the anti-Trop-2 antibody or fragment thereof is the anti-Trop-2 antibody hTINA1 or a fragment thereof; Alternatively, preferably the anti-HER-2 antibody or fragment thereof is the anti-HER-2 antibody trastuzumab or a fragment thereof.

18. 18. A prodrug metabolite produced by the antibody-drug conjugate of any one of claims 15 to 17, wherein the prodrug metabolite has structural formula IX: 【Chemistry 35】 wherein the groups A and CPT have the same meanings as defined in any one of claims 6 to 14 for the groups A and CPT.

19. The compound has structural formula IX-A: 【Transformation 36】 (In structural formula IX-A, groups A, G, Y, R １ , R ２ , R ３ , R ４ , X and n are groups A, G, Y, R １ , R ２ , R ３ , R ４ , X and n have the same meanings as defined in any one of claims 6 to 14, Preferably, the compound represented by structural formula IX-A further comprises a compound represented by structural formula IX-A-1: 【Chemistry 37】 is a compound represented by Alternatively, the compound has structural formula IX-B: 【Transformation 38】 (In structural formula IX-B, groups A, G, R ５ , X and n are groups A, G, R ５ , X and n have the same meanings as defined in any one of claims 6 to 14, Alternatively, the compound has structural formula IX-C: 【Chemistry 39】 (In structural formula IX-C, groups A and R ８ represents groups A and R ８ 19. The prodrug metabolite according to claim 18, characterized in that it is a compound represented by the formula:

20. 20. Use of a compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, or an antibody-drug conjugate, or a prodrug metabolite in the manufacture of a medicament for the treatment of a tumor.

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