Antibody-drug conjugate containing thiazolo[5,4-b]pyridine structure and use thereof

Abstract

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Description

Antibody-drug conjugate containing thiazole [5,4-B] pyridine structure and its use Field of the Invention

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a novel thiol-reactive linker, a linker containing the linker, a linker-payload conjugate, an antibody-drug conjugate based on the linker, and uses thereof, as well as pharmaceutical compositions containing the antibody-drug conjugate, and uses of these antibody-drug conjugates for treating and / or preventing diseases. Background Art

[0002] Antibody-drug conjugates (ADCs) utilize biologically targeted monoclonal antibodies (antibodies) to deliver potent cytotoxins to lesions. Combining the strengths of both antibodies and cytotoxic drugs, they offer numerous advantages, including strong targeting, high activity, minimal toxicity, and a long half-life. Currently, 15 ADCs are marketed globally, with approximately 200 in clinical development.

[0003] ADCs structurally consist of three components: an antibody, a small molecule cytotoxin, and a linker. The antibody's role is to achieve targeting, the cytotoxin's role is to exert its therapeutic effect and kill target cells, and the linker's role is to organically integrate the antibody and cytotoxin into a single, integrated whole. Linker performance is crucial for ensuring targeted delivery of the cytotoxin, directly determining the efficacy, safety, and pharmacokinetic characteristics of the ADC. Therefore, linker construction is crucial for ADC drugs.

[0004] There are many important considerations when developing ADC linkers, including the choice of antibody conjugation site, the average number of cytotoxin molecules conjugated per antibody molecule (drug / antibody ratio, DAR), linker cleavability, and linker hydrophilicity. Fundamental requirements for ADC linkers include ensuring stability in the human circulatory system throughout the therapeutic cycle after administration and rapid and effective release of the payload upon reaching the target tissue. Only in this way can the ADC effectively leverage the dual advantages of antibody targeting and toxin high potency.

[0005] Furthermore, the linker design must match the physicochemical properties of the payload to which it is attached, thereby modulating the hydrophilicity of the linker-payload complex. Appropriate linker hydrophilicity design can enhance the water solubility of the linker-payload complex during the coupling process, improve coupling yield, reduce the degree of polymerization of the product, enhance product stability, and improve product bioavailability and pharmacokinetics.

[0006] Therefore, the choice of linker and conjugation strategy is crucial during the design and development of ADC drugs. The ADC linker must ensure effective connection between the antibody and cytotoxin before reaching the target tissue. If the cytotoxin is released before the ADC reaches the target tissue, the effective payload concentration released by the ADC to the target tissue will be greatly reduced due to the decrease in loading efficiency, thereby reducing the ADC's efficacy. In addition, the detached potent cytotoxin will cause significant off-target toxicity to normal tissues.

[0007] Currently, the vast majority of ADCs currently on the market and in clinical development still utilize the Michael addition reaction between a thiol and a maleimide to form thiosuccinimide (Figure 1). Due to the many advantages of this reaction, such as rapidity, quantitativeness, and mild conditions, no superior alternative has been found in current ADC research.

[0008] However, the sulfosuccinimide groups generated in the ADC products constructed by the above reaction are unstable. After the ADC is administered, it will continuously undergo a retro-Michael reaction in the body, which will cause the highly effective cytotoxin to continuously be released from the ADC and released into normal tissues (Figure 1). The maleimide groups generated by the off-target will further react rapidly with substances containing sulfhydryl groups such as albumin, glutathione, and cysteine ​​in the blood, further accelerating the off-target process of the cytotoxin from the ADC. According to reports, in in vitro mouse plasma, the off-target amount of cytotoxins of ADC can reach as high as 50% within 3 days, and in an in vivo mouse model, the off-target amount of cytotoxins can reach as high as 60% after one week of ADC administration (Nature Biotech., 2014, 32(10):1059-1062.). The above-mentioned ADC metabolism in vivo will undoubtedly lead to reduced ADC efficacy and increased toxicity (Nature Biotech., 2012, 30(2): 184-189.; Bioconjugate Chem., 2008, 19(3): 759-765.; Bioconjug Chem., 2016, 27(7): 1588-1598.; J Med Chem., 2014, 57(19): 7890-7899.).

[0009] Therefore, current mainstream ADC linker technologies are still unable to effectively ensure the full stability of ADCs before reaching target tissues. The construction of next-generation linkers has become a critical issue and major challenge that needs to be addressed in ADC development. In addition to stability, ADCs still have ample room for improvement in many aspects, such as optimizing linker drug release performance, hydrophilicity, and the rational combination of linkers and payloads.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention specifically relates to a novel thiol-reactive linker based on the 2-(methylsulfonyl)thiazolo[5,4-b]pyridine structure (English name: 2-(methylsulfonyl)thiazolo[5,4-b]pyridine), as well as linkers comprising the linker, linker-payload conjugates, antibody-drug conjugates based on such linkers, and uses of the linkers. It also relates to pharmaceutical compositions comprising the antibody-drug conjugates, and uses of these antibody-drug conjugates for treating and / or preventing diseases.

[0012] On the one hand, the present application provides an antibody-drug conjugate, its intermediate, preparation method and application. The antibody-drug conjugate of the present application can realize the widespread application of cytotoxic drugs in the field of ADC, mainly for the treatment of tumor diseases. The main technical effect of the present application is that the linker of the provided novel thiol-reactive linker is generally applicable to the coupling reaction of macromolecules and small molecules containing thiol groups, and is also suitable for coupling various toxins, and the resulting conjugate product has significantly improved stability. For example, when the novel linker in the present application is applied to the field of ADC, it can improve the safety of ADC on the one hand by reducing the off-target of the effective load, and on the other hand, it can improve the efficacy of ADC.

[0013] The reaction between the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine model molecule and the thiol model molecule is as follows:

[0014] A typical preparation method of 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker (core) is as follows:

[0015] Preparation method 1.

[0016] Preparation method 2.

[0017] Preparation method 3.

[0018] Preparation method 4.

[0019] Preparation method 5.

[0020] Preparation method 6.

[0021] Advantages of linkers based on the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine structure include:

[0022] 1. The product after reaction with thiol groups has higher stability than traditional linker products;

[0023] 2. Higher hydrophilicity than traditional connectors;

[0024] 3. Good reaction kinetics with sulfhydryl groups;

[0025] 4. Highly selective reaction with sulfhydryl groups under mild conditions;

[0026] 5. Widely applicable to various biological macromolecules and small molecules containing sulfhydryl groups;

[0027] 6. The raw materials of the linker are easily available, the preparation process is simple, and the reaction yield is high.

[0028] Thus, in one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or isotopic variant thereof:

[0029] in,

[0030] R1 is selected from C 1-6 Alkyl or C 1-6 alkyl halide;

[0031] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NR b -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0032] Where p = 1 or 2;

[0033] L1 is a chemical bond or -(CH2) m1 -(OCH2CH2) n1 -(CH2CH2O) n2 -(CH2) r1 -(L) q -(CH2) r2 -(OCH2CH2) n3 -(CH2CH2O) n4 -(CH2) m2 -;

[0034] wherein -L- is selected from -O-, -NR b -、-C(O)NR b -、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0035] Each of m1, m2, n1, n2, n3, n4, r1 and r2 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0036] q = 0, 1, or 2;

[0037] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0038] R2 is selected from H, D, halogen, -OR a 、-NR b R c or the following groups:

[0039] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0040] s = 0, 1, or 2;

[0041] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and R c and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0042] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0043] In another aspect, the present invention provides use of the compound of formula (I) or a pharmaceutically acceptable salt or isotopic variant thereof in the preparation of an antibody-drug conjugate.

[0044] In another aspect, the present invention provides a compound of formula (II), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof:

[0045] in,

[0046] R1 is selected from C 1-6 Alkyl or C 1-6 alkyl halide;

[0047] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0048] s = 0, 1, or 2;

[0049] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NR b -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0050] Where p = 1 or 2;

[0051] L1 is a chemical bond or -(CH2) m -(OCH2CH2) n -(CH2CH2O) n -(CH2) r -(L) q -(CH2) r -(OCH2CH2) n -(CH2CH2O) n -(CH2) m -;

[0052] wherein -L- is selected from -O-, -NR b -、-C(O)NR b -、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0053] Each m, n, r is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0054] q = 0, 1, or 2;

[0055] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0056] L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4;

[0057] The N-terminus of the amino acid residue or oligopeptide residue is connected to W2, and the C-terminus is connected to L3;

[0058] R4 is selected from D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl, C 1-6 Halogenated alkyl, polyethylene glycol, polysarcosine, pentose, hexose, sulfonic acid group, methylsulfonyl group, phosphate group, phosphite group, quaternary ammonium salt, or selected from the following groups:

[0059] L3 is selected from the following structures:

[0060] Among them, NH is connected to L2, and C(O) is connected to D;

[0061] R5 is selected from H, D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl or C 1-6 alkyl halide;

[0062] t = 0, 1, 2, 3, or 4;

[0063] R6 is selected from H,

[0064] D is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors;

[0065] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and R c and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0066] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0067] In another aspect, the present invention provides a compound of formula (III), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, which has the following general formula:

[0068] in,

[0069] A is a targeting molecule;

[0070] x = 1, 2, 3, 4, 5, 6, 7, or 8;

[0071] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0072] s = 0, 1, or 2;

[0073] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NR b -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0074] Where p = 1 or 2;

[0075] L1 is a chemical bond or -(CH2) m -(OCH2CH2) n -(CH2CH2O) n -(CH2) r -(L) q -(CH2) r -(OCH2CH2) n -(CH2CH2O) n -(CH2) m -;

[0076] wherein -L- is selected from -O-, -NR b -、-C(O)NR b-、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0077] Each m, n, r is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0078] q = 0, 1, or 2;

[0079] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0080] L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4;

[0081] The N-terminus of the amino acid residue or oligopeptide residue is connected to W2, and the C-terminus is connected to L3;

[0082] R4 is selected from D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl, C 1-6 Halogenated alkyl, polyethylene glycol, polysarcosine, pentose, hexose, sulfonic acid group, methylsulfonyl group, phosphate group, phosphite group, quaternary ammonium salt, or selected from the following groups:

[0083] L3 is selected from the following structures:

[0084] Among them, NH is connected to L2, and C(O) is connected to D;

[0085] R5 is selected from H, D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl or C 1-6 alkyl halide;

[0086] t = 0, 1, 2, 3, or 4;

[0087] R6 is selected from H,

[0088] D is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors;

[0089] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and R c and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0090] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0091] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, and a pharmaceutically acceptable excipient or adjuvant.

[0092] In another aspect, the present invention provides the use of the compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof in the preparation of a medicament for treating a disease or condition, and alleviating the severity of the disease or condition.

[0093] In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso-racemate, racemate or tautomer thereof, or a pharmaceutical composition of the present invention for use in treating a disease or condition, and alleviating the severity of the disease or condition.

[0094] In another aspect, the present invention provides a method for treating a disease or condition in a subject, and alleviating the severity of the disease or condition, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso-racemate, racemate or tautomer thereof, or a pharmaceutical composition of the present invention.

[0095] On the other hand, the present application provides a method for preparing an antibody-drug conjugate, wherein the disulfide chain in the hinge region of the antibody or antibody fragment is reduced to generate a pair of cysteine ​​residues, and the sulfhydryl group in the cysteine ​​residue is substituted with the linker of the compound of the present invention, and then the compound of the present invention is linked to the cysteine ​​sulfhydryl group of the antibody or antibody fragment to obtain an antibody-drug conjugate. The drug-antibody coupling ratio (DAR) can be controlled according to the reaction conditions, for example, it is commonly between 2 and 8.

[0096] On the other hand, DAR (i.e., the average molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after conjugation of a single monoclonal antibody molecule to a cytotoxic drug) can generally be measured by hydrophobic-interaction chromatography (HIC), reverse phase high performance liquid chromatography (RP-HPLC), polyacrylamide-SDS gel electrophoresis (SDS PAGE, electrophoresis), liquid chromatograph-mass spectrometer (LC-MS), ultraviolet / visible spectroscopy (UV / Vis), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1: Diagram of maleimide-linker-based ADC construction and toxin off-target reactions of the product.

[0098] Figure 2: Stability study of the ADC of the present invention in human plasma.

[0099] Figure 3: Stability study of the ADC of the present invention in mouse plasma.

[0100] Figure 4: In vitro cytotoxicity study of MMAE, Dxd, Compound-41 and Staurosporine.

[0101] Figure 5: In vivo efficacy study of ADC-14B.

[0102] Figure 6: Comparative efficacy study of ADC-14B, DS7300-43, and IgG-14B.

[0103] Figure 7: In vivo efficacy study of HDC-14E ​​(DAR4).

[0104] Detailed Description of the Invention

[0105] definition

[0106] Chemical definition

[0107] Definitions of specific functional groups and chemical terms are described in more detail below.

[0108] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0109] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl is preferred. In some embodiments, C 1-2 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen (O), sulfur (S), nitrogen (N), boron (B), silicon (Si), phosphorus (P)). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0110] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0111] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-3 Halogenated alkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0112] “C 2-6 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). In some embodiments, C 2-4 Alkenyl is particularly preferred. Examples of the alkenyl include, but are not limited to, vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Regardless of whether the alkenyl is modified with "substituted", each of the alkenyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0113] “C 2-6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). In some embodiments, C 2-4 Alkynyl is particularly preferred. In some embodiments, alkynyl does not contain any double bond. One or more carbon triple bonds can be internal (e.g., in 2-butynyl) or end (e.g., in 1-butynyl). Examples of the alkynyl include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. Regardless of whether "substituted" is modified before the alkynyl, each of the alkynyl groups is optionally substituted independently, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined as follows.

[0114] “C 3-10"Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-8 Cycloalkyl, C 4-6 Cycloalkyl is preferred, C 3-6 Cycloalkyl is particularly preferred, more preferably C 5-6 Cycloalkyl. Cycloalkyl also includes ring systems in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), cyclononyl (C9), cyclononenyl (C9), cyclodec ... 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ), bornyl, adamantyl, etc. Regardless of whether the cycloalkyl group is preceded by "substituted", each of the cycloalkyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0115] “C 3-8 "Cycloalkylene" refers to the group with "C 3-8 A divalent group is formed by another hydrogen atom of the "cycloalkyl".

[0116] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. In some embodiments, 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 5-10 membered heterocyclyl is preferred, which is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl is preferred, which is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, 3-6 membered heterocyclyl is particularly preferred, which is a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably, 5-6 membered heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl, aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Regardless of whether the heterocyclyl group is preceded by "substituted", each of the heterocyclyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents or 1 substituent, suitable substituents being defined below.

[0117] The "3- to 8-membered heterocyclic group" refers to a divalent group formed by removing another hydrogen atom of the "3- to 8-membered heterocyclic group".

[0118] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thietidinyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolane, oxasulfuranyl, disulfuranyl, and Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithian ... Alkyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxepanyl, and thiecanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to, dihydroindole, isoindole, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzo Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclyl groups) fused to a C6 aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0119] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C 10 "aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, C 6-10 Aryl is particularly preferred, with C6 aryl being more preferred. Aryl also includes ring systems in which the aforementioned aryl ring is fused to one or more cycloalkyl or heterocyclic groups, with the point of attachment being on the aryl ring. In this case, the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Regardless of whether the aryl group is preceded by "substituted," each aryl group is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Suitable substituents are defined below.

[0120] “C 6-10 "Arylene" refers to removing "C 6-10 The other hydrogen atom of the "aryl" forms a divalent group.

[0121] "5-10 membered heteroaryl" refers to a group of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl rings are fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5-membered heteroaryl is particularly preferred, which is a 5-membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Regardless of whether the heteroaryl group is preceded by "substituted", each of the heteroaryl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

[0122] The "5- to 10-membered heteroarylene group" refers to a divalent group formed by removing another hydrogen atom of the "5- to 10-membered heteroarylene group".

[0123] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepine, oxepinyl, and thiepine. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0124] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group can but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0125] Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2, -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa 、-NRbb SO2R aa 、-SO2N(R bb )2, -SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2, -C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0126] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb)2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0127] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0128] R bb Each of the following is independently selected from: hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0129] R ccEach of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0130] R dd Each of the is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2,、-N(R ff )3 + X - 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2R ee 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0131] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0132] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0133] R gg Each of the independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - 、-NH(C 1-6 Alkyl)2 + X - 、-NH2(C 1-6 alkyl) + X - 、-NH3+ X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 Alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic group, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0134] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom ccThe groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substituted, and wherein R aa 、R bb 、R cc and R dd As mentioned above.

[0135] "Deuterated" or "deuterium" refers to a compound or group in which one or more hydrogen atoms are replaced by deuterium; deuteration can be mono-, di-, poly-, or per-substituted. The terms "one or more deuterated" and "one or more deuterated" are used interchangeably. The deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, and even more preferably greater than 99%.

[0136] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 1000 or more than 10000. In this application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by a chemical bond, and the biomacromolecule may be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000.

[0137] As used herein, the term "compound of the present application" refers to the compound of the present application. The term also includes various pharmaceutically acceptable salts, prodrugs, hydrates, solvates, enantiomers, diastereomers, meso-, racemic- or tautomers of the compound of the present application.

[0138] Biological definition

[0139] In this application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to cell-associated antigens or receptors. The function of the ligand can be to present the drug to the cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells, receptors and / or antigens. In this application, the ligand can be expressed as Ab, and the ligand antigen forms a connection bond with the connecting unit through the heteroatom on the ligand, which can be an antibody or an antigen-binding fragment thereof. The antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody or an antigen-binding fragment thereof that targets a target selected from the following groups:

[0140] HER2, Trop-2, Claudin-6, Claudin-9, Claudin-18.2, EGFR, c-Met, CD19, PSMA, Muc1, BCMA, PD-L1, CD33, CD30, CD22, CD79b, Nectin-4, CD19, tissue factor, FRα, B7-H3, B7-H4, CDH3, CDH6, CDH17, ALPP, CD56, CD37, HER3, ROR1, MSLN, TNF-α, CD25, ENPP 3. Muc1, Axl, CD20, ROR2, GPNMB, CEACAM5, CEACAM6, CD138, GC-C, LIV-1, CA6, FUT3, IGF-1R, CTLA4, RNF43, DPEP3, 5T4, I TGB6, EFNA4, CD228, Notch3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, TIM1, sTn, ETB, Globo H, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD5 1, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, LYFD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, Muc1, LRRC15, CD44, CD70, EphA2, CXCR4, DDR1, DKL1, FOLR, CD45, DSG2, ALK, TRA1L, EpCAM, VEGFR2, CD47, CD49, SSEA-4, DCLK1, OacGD2, CD73, ENO1, BSG, CD24, GLUT1 and GPRC5D, preferably HER2, HER3, EGFR, Trop-2, Claudin-6, Claudin-18.2, B7-H3, B7-H4, CDH3, CDH6, CDH17, FRα, ROR1, ALPP, CEACAM5, CEACAM6 or FOLR.

[0141] In the present application, the term "conjugate" generally refers to a compound prepared by one or more chemical reactions of the compounds of the present application, or connected to each other through one or more connecting structures such as a bridge, a spacer, or a connecting part.

[0142] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2), for example, the term "HER2" refers to any natural HER2 from any human source. The term also encompasses "full-length" and unprocessed HER2 and any form of HER2 (e.g., mature protein) derived from processing in a cell. The term also encompasses naturally occurring variants and isoforms of HER2, such as splice variants or allelic variants. For example, Uniprot accession number P04626 provides a description of HER2 and sequences.

[0143] In this application, the terms "B7-H3" and "CD276" generally refer to a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. The B7-H3 protein is encoded by a gene on chromosome 15q24 and consists of 316 amino acids encompassing an extracellular domain, a transmembrane domain, and a short intracellular domain. The intracellular domain of the B7-H3 protein is very short and has no known signaling motif.

[0144] In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To create a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody can be established. The variable region genes can then be cloned from the murine hybridoma cells. The constant region genes of a human antibody can then be cloned as needed. The murine variable region genes and the human constant region genes can then be linked to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in either eukaryotic or prokaryotic systems.

[0145] In this application, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to antibodies produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.

[0146] In this application, the terms "fully human antibody", "fully human antibody" or "completely human antibody" are also called "fully human monoclonal antibody", and the variable region and constant region of the antibody can be both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0147] In the present application, term " CDR " generally refers to one of 6 hypervariable regions that mainly contribute to antigen binding in the variable domains of antibody.One of the most commonly used definitions of the 6 CDRs is provided by Kabat EA et al., Chothia et al. and MacCallum et al. As used in the present application, the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 (CDRL1, CDRL2, CDRL3 or LK L2, L3) of light chain variable domain, and CDRK, CDR2 and CDR3 (CDRH1, CDRH2, CDRH3 or H1, H2, H3) of heavy chain variable domain.

[0148] In this application, the term "linker" generally refers to a chemical structure fragment or bond that is connected to one group at one end and to another group at the other end. It can also be connected to other linkers and then connected to the drug and / or ligand. The direct or indirect connection to the ligand can mean that the group is directly connected to the ligand through a covalent bond, or it can be connected to the ligand through a linker. For example, a chemical structure fragment or bond containing an acid-labile linker structure (such as a hydrazone), a protease-sensitive (such as a peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as a linker.

[0149] In this application, the term "linking group" generally refers to a group capable of connecting to another group. For example, a compound having a linking group can be connected to another group through a coupling reaction between the linking group and the other group. For example, a maleimide group can serve as a linking group.

[0150] In this application, the term "Drug unit" generally refers to a chemical moiety that is directly or indirectly conjugated to an antibody or antigen-binding fragment to form an immunoconjugate. For example, a "Drug unit" includes, but is not limited to, a compound with anti-tumor activity as described herein. For example, a Drug unit includes a topoisomerase inhibitor.

[0151] In this application, the term "disease associated with the expression of a target" generally means that the occurrence and / or progression of the disease is associated with the expression level of the target. For example, the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is increased relative to the expression level in normal cells from the tissue or organ, i.e., high expression. Or, for example, the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is decreased relative to the expression level in normal cells from the tissue or organ, i.e., low expression. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, express a target, i.e., positive. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, do not express a target, i.e., negative. For example, the characteristics of target expression can be determined by standard assays known in the art.

[0152] As used herein, "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric antibodies or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens generally have a large number of binding sites, also known as epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen or a portion thereof that specifically binds to a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins described herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulins are derived from humans, mice, or rabbits. "Antibody fragments" can comprise a portion of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include: Fab, Fab\F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies; fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity determining regions); and any of the above-mentioned epitope-binding fragments that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The antibodies constituting the antibody-drug conjugates herein can retain their antigen-binding ability in their original wild-type state. Therefore, the antibodies in the present application can, for example, specifically bind to antigens. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as known or predicted functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known antibodies). The antigens bound by antibodies can be one or a subset of the above categories, while other subsets contain other molecules / antigens with special properties (compared to the target antigen). The antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared by antibody preparation methods and information well known in the industry.These targets can be specifically expressed on the surface of one or more cancer cells, but rarely or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides can be more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells.

[0153] In this application, the term "amino acid" refers to the basic structural unit of proteins and is the basis for later protein modification in organisms. There are a total of 20 natural amino acids. In addition, based on these basic amino acids, organisms will also synthesize amino acid types derived from hydroxyproline, hydroxylysine, etc. In fireflies, they can even synthesize D-amino acids. Natural amino acids are generally L-type, and the present invention also includes D-type amino acids. The amino acids used in the present invention are shown in the following table:

[0154] In the present application, the term "polypeptide residue / oligopeptide residue" generally refers to a residue comprising two or more amino acid residues linked by peptide bonds. For example, two or more amino acids in a polypeptide residue may be optionally substituted. For example, the polypeptide residue of the present application may be selected from the following groups: valine-citrulline (Val-Cit), valine-alanine (Val-Ala), valine-lysine (Val-Lys), phenylalanine-lysine (Phe-Lys), lysine-lysine (Lys-Lys), alanine-lysine (Ala-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ileucine-Isosorbide-5-Nitrae-D-Yeast), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Isosorbide-5-Nitrae-D-Yeast), le ... le-Cit), Phenylalanine-Alanine (Phe-Ala), Lysine-Valine-Citrulline (Lys-Val-Cit), Lysine-Valine-Alanine (Lys-Val-Ala), Valine-Lysine-Glycine (Val-Lys-Gly), Glycine-Valine-Lysine (Gly-Val-Lys), Glycine-Valine-Alanine (Gly-Val-Ala), Glutamic Acid-Valine-Alanine (Glu-Val-Ala), al-Ala), Glutamic acid-Valine-Citrulline (Glu-Val-Cit), Glutamine-Valine-Ala (Gln-Val-Ala), Glutamine-Valine-Citrulline (Gln-Val-Cit), Alanine-Alanine-Ala (Ala-Ala-Ala), Alanine-Alanine-Asparagine (Ala-Ala-Asn), Phenylalanine-Phenylalanine-Lysine (Phe-Phe-Lys), Glycine- Phenylalanine-lysine (Gly-Phe-Lys), Leucine-Alanine-Leucine (Leu-Ala-Leu), Isoleucine-Alanine-Leucine (Ile-Ala-Leu), Valine-Alanine-Valine (Val-Ala-Val), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), (Ala-Leu-Ala-Leu) and (Gly-Phe-Leu-Gly),Preferably selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamine-valine-alanine (Gln-Val-Ala), glutamine-valine-citrulline (Gln-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), preferably selected from the group consisting of valine-citrulline (Val-Cit), valine-alanine (Val-Ala), valine- Lysine (Val-Lys), Phenylalanine-Lysine (Phe-Lys), Lysine-Valine-Citrulline (Lys-Val-Cit), Lysine-Valine-Alanine (Lys-Val-Ala), Glutamine-Valine-Alanine (Gln-Val-Ala), Glutamine-Valine-Citrulline (Gln-Val-Cit) and Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), preferably selected from the following group: Valine-Citrulline (Val-Cit), Valine-Alanine (Val-Ala), Valine-Lysine (Val-Lys), Lysine-Valine-Citrulline (Lys-Val-Cit), Lysine-Valine-Alanine (Lys-Val-Ala) and Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly).

[0155] In this application, the term "polyethylene glycol group / polyethylene glycol group" generally refers to a residue comprising one or more ethylene glycol residues connected together. For example, a polyethylene glycol group may comprise -(CH2CH2O) p -, wherein p is a number of at least 1. For example, the polyethylene glycol group in the present application may be optionally substituted.

[0156] In this application, the term "polysarcosine residue / polysarcosine residue" generally refers to a residue comprising one or more sarcosine residues linked together. For example, a polysarcosine residue may comprise -(COCH2N(CH3)) q -, where q is a number at least 1.

[0157] In this application, the term "pentose" refers to a monosaccharide with five carbon atoms in its molecule, also known as a pentose. Pentose is widely distributed in the biological world and plays an important role in life activities. The main ones are D-xylose, L-arabinose, D-ribose and its derivative D-2-deoxyribose. Ketopentose, which are intermediates in sugar metabolism, include D-ribulose and D-xylulose.

[0158] In this application, the term "hexose" refers to a monosaccharide containing 6 carbon atoms, also known as a hexose. Hexoses are the most widely distributed in nature, have the largest number, and are most closely related to the body's nutritional metabolism. A 6-carbon sugar containing an aldehyde group at position 1 is called an aldohexose, and a hexose containing a ketone group at position 2 is called a ketohexose. Five of the six carbon atoms in a hexose are connected to a hydroxyl group (-OH), and the remaining carbon atom belongs to an aldehyde group (-COH) or a ketone group (-CO-). Important aldohexoses include D-glucose, D-galactose, and D-mannose; ketohexoses include D-fructose.

[0159] In this application, the term "tumor" generally refers to any new, pathological tissue proliferation. For purposes of this application, angiogenesis is part of the characteristic features of tumors. Tumors may be benign or malignant. The term "tumor" is generally used to refer to benign or malignant tumors, while the term "cancer" is generally used to refer to malignant tumors, which may be metastatic or non-metastatic. Tumors treatable by the methods of this application are selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma. For research purposes, these tissues can be isolated from readily available sources using methods well known to those skilled in the art.

[0160] Other definitions

[0161] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0162] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0163] As used herein, the term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt of the compounds of the invention which are suitable for use in contact with the tissues of patients, within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including, where possible, zwitterionic forms of the compounds of the invention.

[0164] Pharmaceutically acceptable salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, borates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, and the like, prepared from inorganic acids. Representative salts include hydrobromides, hydrochlorides, sulfates, bisulfates, nitrates, borates, and phosphates. Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Representative salts include acetate, propionate, valerate, oleate, palmitate, stearate, laurate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, glucoheptonate, lactobionate, laurylsulfonate, and isethionate, etc. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Also contemplated are salts of amino acids, such as argininate, gluconate, galacturonate, and the like (see, e.g., Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0165] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those of the general formulae or specific compounds described herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example, 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O.31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium (i.e. 2 H) substitution may be preferred in some cases because greater metabolic stability may provide therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when performing the processes disclosed in the following schemes and / or the Examples and Preparations.

[0166] The compounds of the present invention include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0167] As used herein, the term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical compositions can be prepared using techniques commonly used in the art.

[0168] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or polypeptides, genes and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. For example, a pharmaceutically acceptable carrier can be distinguished from a nucleic acid vector used to contain a gene in genetic engineering. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.

[0169] As used herein, the term "compound with anti-tumor activity" generally refers to a compound that has the ability to reduce the proliferation rate, viability, or metastatic activity of tumor cells. For example, anti-tumor activity can be demonstrated by a decrease in the growth rate of abnormal cells or a stabilization or reduction in tumor size during treatment, or by a prolonged survival period resulting from treatment compared to a control without treatment. Anti-tumor activity can be assessed using recognized in vitro or in vivo tumor models, such as xenograft models.

[0170] In some embodiments of the present invention, the biologically active molecule in the conjugate is a compound with specific anti-tumor activity, such as: radioactive isotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 or radioactive isotopes of Lu; metal complexes, such as metal platinum complexes (such as oxaliplatin) or metal gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; topoisomerase inhibitors; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nelarabine; drugs that act on structural proteins, such as microtubule inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatin, Tubulysin B or eribulin, etc.); tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors; epigenetic-related target inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors.

[0171] In this application, the term "topoisomerase inhibitor" generally refers to compounds including topoisomerase I inhibitors and topoisomerase II inhibitors or their derivatives. Examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs; topoisomerase II inhibitors (such as actinomycin D, doxorubicin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide, etc.). Topoisomerase can refer to an enzyme that corrects the number of DNA loops by cutting the phosphodiester bonds in one or both strands of DNA and then rewinding and sealing.

[0172] In this application, the term "camptothecin analogue" generally refers to a compound that is structurally similar to or derived from camptothecin. For example, the structure of camptothecin can be recorded in CAS accession number 7689-03-4. For example, a camptothecin analogue can refer to isatecan (CAS accession number 171335-80-1) or belotecan (CAS accession number 256411-32-2). The term "non-camptothecin topoisomerase I inhibitor" generally refers to heterocyclic molecules with topoisomerase I inhibitory activity of indolecarbazoles, indenoisoquinolinones, benzophenanthridines, and dibenzonaphthyridinones, primarily referring to Genz-644282 (CAS accession number 529488_28-6).

[0173] As used herein, the term "effective amount" generally refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a given subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation, which is within the judgment of the clinician.

[0174] The term "patient" includes both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, and including unborn mammals. Examples of non-mammals include, but are not limited to, birds, fish, and the like.

[0175] The term "patient" includes confirmed patients, but the "patient" does not need to have any special identity with respect to the hospital, clinic or research facility (such as being a confirmed patient, research participant, etc.).

[0176] In this application, the term "hydrophobic chromatography" generally refers to an analytical technique based on differences in the hydrophobicity of substances.

[0177] In this application, the term "liquid chromatography-mass spectrometry" generally refers to an analytical method for identifying the components of a substance. For example, liquid chromatography-mass spectrometry can be used to analyze the molecular weight of a substance to be tested by liquid chromatography-mass spectrometry. Specific implementation plan

[0178] In one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or isotopic variant thereof:

[0179] in,

[0180] R1 is selected from C 1-6 Alkyl or C 1-6 alkyl halide;

[0181] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NR b -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0182] Where p = 1 or 2;

[0183] L1 is a chemical bond or -(CH2) m1 -(OCH2CH2) n1 -(CH2CH2O) n2 -(CH2) r1 -(L) q -(CH2) r2 -(OCH2CH2) n3 -(CH2CH2O) n4 -(CH2) m2 -;

[0184] wherein -L- is selected from -O-, -NR b -、-C(O)NR b -、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0185] Each of m1, m2, n1, n2, n3, n4, r1 and r2 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0186] q = 0, 1, or 2;

[0187] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0188] R2 is selected from H, D, halogen, -OR a 、-NR b R c or the following groups:

[0189] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0190] s = 0, 1, or 2;

[0191] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and R c and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0192] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0193] In another aspect, the present invention provides a compound of formula (II), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof:

[0194] in,

[0195] R1 is selected from C 1-6 Alkyl or C 1-6 alkyl halide;

[0196] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0197] s = 0, 1, or 2;

[0198] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NRb -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0199] Where p = 1 or 2;

[0200] L1 is a chemical bond or -(CH2) m -(OCH2CH2) n -(CH2CH2O) n -(CH2) r -(L) q -(CH2) r -(OCH2CH2) n -(CH2CH2O) n -(CH2) m -;

[0201] wherein -L- is selected from -O-, -NR b -、-C(O)NR b -、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0202] Each m, n, r is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0203] q = 0, 1, or 2;

[0204] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0205] L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4;

[0206] The N-terminus of the amino acid residue or oligopeptide residue is connected to W2, and the C-terminus is connected to L3;

[0207] R4 is selected from D, halogen, NO2, -OR a 、-NR b Rc 、C 1-6 Alkyl, C 1-6 Halogenated alkyl, polyethylene glycol, polysarcosine, pentose, hexose, sulfonic acid group, methylsulfonyl group, phosphate group, phosphite group, quaternary ammonium salt, or selected from the following groups:

[0208] L3 is selected from the following structures:

[0209] Among them, NH is connected to L2, and C(O) is connected to D;

[0210] R5 is selected from H, D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl or C 1-6 alkyl halide;

[0211] t = 0, 1, 2, 3, or 4;

[0212] R6 is selected from H,

[0213] D is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors;

[0214] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and R c and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0215] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0216] In another aspect, the present invention provides a compound of formula (III), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, which has the following general formula:

[0217] in,

[0218] A is a targeting molecule;

[0219] x = 1, 2, 3, 4, 5, 6, 7, or 8;

[0220] R3 is selected from H, D, halogen, C 1-6 Alkyl or C 1-6 alkyl halide;

[0221] s = 0, 1, or 2;

[0222] W1 is selected from -O-, -S-, -NR b -、-C(O)O-、-C(O)NR b -、-OC(O)-、-NR b -C(O)-, -S(O) p O-or-OS(O) p -;

[0223] Where p = 1 or 2;

[0224] L1 is a chemical bond or -(CH2) m -(OCH2CH2) n -(CH2CH2O) n -(CH2) r -(L) q -(CH2) r -(OCH2CH2) n -(CH2CH2O) n -(CH2) m -;

[0225] wherein -L- is selected from -O-, -NR b -、-C(O)NR b -、-C(O)O-、-NR b -C(O)-, -OC(O)-, -C 3-8 Cycloalkylene-, -3-8 membered heterocyclylene-, -C 6-10 Arylene- or -5- to 10-membered heteroarylene;

[0226] Each m, n, r is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0227] q = 0, 1, or 2;

[0228] W2 is selected from chemical bonds, -O-, -S-, -NR b -or-C(O)-;

[0229] L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4;

[0230] The N-terminus of the amino acid residue or oligopeptide residue is connected to W2, and the C-terminus is connected to L3;

[0231] R4 is selected from D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl, C 1-6 Halogenated alkyl, polyethylene glycol, polysarcosine, pentose, hexose, sulfonic acid group, methylsulfonyl group, phosphate group, phosphite group, quaternary ammonium salt, or selected from the following groups:

[0232] L3 is selected from the following structures:

[0233] Among them, NH is connected to L2, and C(O) is connected to D;

[0234] R5 is selected from H, D, halogen, NO2, -OR a 、-NR b R c 、C 1-6 Alkyl or C 1-6 alkyl halide;

[0235] t = 0, 1, 2, 3, or 4;

[0236] R6 is selected from H,

[0237] D is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors;

[0238] where R a 、R b and R c Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl; or R b and Rc and the nitrogen atom to which they are attached together form a 3-10 membered heterocyclic group;

[0239] The above groups are optionally substituted by one or more deuterium groups, up to full deuteration.

[0240] A

[0241] In one embodiment, A is a targeting molecule; in a specific embodiment, A is selected from a protein, an antibody, an antibody fragment, a fusion protein, a polypeptide, an enzyme and a small molecule; in one embodiment, A is a protein; in another embodiment, A is an antibody, preferably a monoclonal antibody, such as a monospecific monoclonal antibody and a bispecific monoclonal antibody; in another embodiment, A is an IgG antibody and an HCAb antibody targeting a tumor-associated antigen; in one embodiment, A is an antibody fragment; in one embodiment, A is a fusion protein; in one embodiment, A is a polypeptide; in one embodiment, A is an enzyme; in one embodiment, A is a small molecule.

[0242] x

[0243] In one embodiment, x=1; in another embodiment, x=2; in another embodiment, x=3; in another embodiment, x=4; in another embodiment, x=5; in another embodiment, x=6; in another embodiment, x=7; in another embodiment, x=8; in another embodiment, x=2, 3, 4, 5, 6, 7 or 8; in another embodiment, x=2, 4, 6 or 8; in another embodiment, x=4, 6 or 8.

[0244] R3

[0245] In one embodiment, R3 is H; in another embodiment, R3 is D; in another embodiment, R3 is halogen; in another embodiment, R3 is C 1-6 Alkyl; in another embodiment, R3 is C 1-6 Halogenated alkyl.

[0246] s

[0247] In one embodiment, s=0; in another embodiment, s=1; in another embodiment, s=2.

[0248] W1

[0249] In one embodiment, W1 is -O-; in another embodiment, W1 is -S-; in another embodiment, W1 is -NR b-; In another embodiment, W1 is -C(O)O-; In another embodiment, W1 is -C(O)NR b -; In another embodiment, W1 is -OC(O)-; In another embodiment, W1 is -NR b -C(O)-; In another embodiment, W1 is -S(O) p O-; in another embodiment, W1 is -OS(O) p -.

[0250] In one embodiment, p=1; in another embodiment, p=2.

[0251] L1

[0252] In one embodiment, L1 is a chemical bond; in another embodiment, L1 is -(CH2) m1 -(OCH2CH2) n1 -(CH2CH2O) n2 -(CH2) r1 -(L) q -(CH2) r2 -(OCH2CH2) n3 -(CH2CH2O) n4 -(CH2) m2 -.

[0253] In another embodiment, L1 is -(CH2) m1 -(CH2CH2O) n1 -(CH2) r1 -L-(CH2) r2 -(CH2CH2O) n2 -(CH2) m2 -; In another embodiment, L1 is -(CH2) m3 -(OCH2CH2) n3 -(CH2) r3 -L-(CH2) r4 -(OCH2CH2) n4 -(CH2) m4 -.

[0254] In another embodiment, L1 is -(CH2CH2O) n5 -(CH2) r5 -L-(CH2) m5 -; In another embodiment, L1 is -(OCH2CH2) n6 -(CH2) r6 -L-(CH2) m6-; In another embodiment, L1 is -(CH2) m7 -L-(CH2CH2O) n7 -(CH2) r7 -; In another embodiment, L1 is -(CH2) m8 -L-(OCH2CH2) n8 -(CH2) r8 -.

[0255] In another embodiment, L1 is -(CH2) m9 -(CH2CH2O) n9 -(CH2) r9 -; In another embodiment, L1 is -(CH2) m10 -(OCH2CH2) n10 -(CH2) r10 -.

[0256] In another embodiment, L1 is -(CH2CH2O) n11 -(CH2) m11 -; In another embodiment, L1 is -(CH2) m12 -(CH2CH2O) n12 -; In another embodiment, L1 is -(OCH2CH2) n13 -(CH2) m13 -; In another embodiment, L1 is -(CH2) m14 -(OCH2CH2) n14 -.

[0257] In another embodiment, L1 is -(CH2) m15 -O-(CH2) m16 -; In another embodiment, L1 is -(CH2) m17 -O-, -(CH2) m18 -NH-; In another embodiment, L1 is -O-(CH2) m19 -; In another embodiment, L1 is -NR b -(CH2) m20 -; In another embodiment, L1 is -(CH2) m21 -; in another embodiment, L1 is -L-; in another embodiment, L1 is -(CH2) m22 -L-; In another embodiment, L1 is -L-(CH2) m23 -.

[0258] In another embodiment, L1 is -(CH2CH2O) n11-(CH2) m11 -, preferably -(CH2CH2O) n11 -; In another embodiment, L1 is -(CH2) m21 -.

[0259] In one embodiment, -L- is -O-; in another embodiment, -L- is -NR b -; In another embodiment, -L- is -C(O)NR b -; in another embodiment, -L- is -C(O)O-; in another embodiment, -L- is -NR b -C(O)-; in another embodiment, -L- is -OC(O)-; in another embodiment, -L- is -C 3-8 In another embodiment, -L- is -3-8 membered heterocyclylene-; In another embodiment, -L- is -C 6-10 Arylene-; In another embodiment, -L- is -5-10 membered heteroarylene.

[0260] In one embodiment, m=0; in another embodiment, m=1; in another embodiment, m=2; in another embodiment, m=3; in another embodiment, m=4; in another embodiment, m=5; in another embodiment, m=6; in another embodiment, m=7; in another embodiment, m=8; in another embodiment, m=9; in another embodiment, m=10.

[0261] In one embodiment, n=0; in another embodiment, n=1; in another embodiment, n=2; in another embodiment, n=3; in another embodiment, n=4; in another embodiment, n=5; in another embodiment, n=6; in another embodiment, n=7; in another embodiment, n=8; in another embodiment, n=9; in another embodiment, n=10.

[0262] In one embodiment, r=0; in another embodiment, r=1; in another embodiment, r=2; in another embodiment, r=3; in another embodiment, r=4; in another embodiment, r=5; in another embodiment, r=6; in another embodiment, r=7; in another embodiment, r=8; in another embodiment, r=9; in another embodiment, r=10.

[0263] In one embodiment, q=0; in another embodiment, q=1; and in another embodiment, q=2.

[0264] In another specific embodiment, m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, m12, m13, m14, m15, m16, m17, m18, m19, m20, m21, m22, m23 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n1, n2, n3, n4, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r1, r2, r3, r4, r5, r6, r7, r8, r9, r10 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0265] W2

[0266] In one embodiment, W2 is a chemical bond; in another embodiment, W2 is -O-; in another embodiment, W2 is -S-; in another embodiment, W2 is -NR b -; In another embodiment, W2 is -C(O)-.

[0267] L2

[0268] In one embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted with 1, 2, 3, 4, 5 or 6 R4; in another embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-8 amino acids, wherein The amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4; in another embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-6 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine , alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4; in another embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-5 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline , citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4; in another embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-4 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4.

[0269] In one embodiment, L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-5 amino acids, wherein the amino acid is selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R4.

[0270] In another embodiment, L2 is selected from the dipeptide, tripeptide or tetrapeptide residues consisting of the following amino acids, wherein the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6 R4.

[0271] In another embodiment, L2 is selected from the group consisting of valine-citrulline, valine-alanine, valine-lysine, phenylalanine-lysine, lysine-lysine, alanine-lysine, phenylalanine-citrulline, leucine-citrulline, isoleucine-citrulline, phenylalanine-alanine, lysine-valine-citrulline, lysine-valine-alanine, valine-lysine-glycine, glycine-valine-lysine, glycine-valine-alanine, glutamine-valine-alanine, glutamine-valine-citrulline, glutamine- R4 is optionally substituted with 1, 2, 3, 4, 5 or 6 R4.

[0272] In another specific embodiment, L2 is selected from glutamine-valine-alanine, glutamine-valine-citrulline, valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine, and the amino acids are optionally substituted with 1, 2, 3, 4, 5 or 6 R4.

[0273] In another specific embodiment, L2 is selected from valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine, and said amino acids are optionally substituted with 1, 2, 3, 4, 5 or 6 R4.

[0274] R4

[0275] In one embodiment, R4 is D; in another embodiment, R4 is halogen; in another embodiment, R4 is NO2; in another embodiment, R4 is -OR a In another embodiment, R4 is -NR b R cIn another embodiment, R4 is C 1-6 Alkyl; in another embodiment, R4 is C 1-6 In another embodiment, R4 is polyethylene glycol; In another embodiment, R4 is polysarcosine; In another embodiment, R4 is a pentose; In another embodiment, R4 is a hexose; In another embodiment, R4 is a sulfonic acid group; In another embodiment, R4 is a methylsulfonyl group; In another embodiment, R4 is a phosphate group; In another embodiment, R4 is a phosphite group; In another embodiment, R4 is a quaternary ammonium salt; In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is In another embodiment, R4 is

[0276] L3

[0277] In one embodiment, L3 is In another embodiment, L3 is In another embodiment, L3 is

[0278] R5

[0279] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is halogen; in another embodiment, R5 is NO2; in another embodiment, R5 is -OR a In another embodiment, R5 is -NR b R c In another embodiment, R5 is C 1-6 Alkyl; in another embodiment, R5 is C 1-6 Halogenated alkyl.

[0280] t

[0281] In one embodiment, t=0; in another embodiment, t=1; in another embodiment, t=2; in another embodiment, t=3; in another embodiment, t=4.

[0282] R6

[0283] In one embodiment, R6 is H; in another embodiment, R6 is In another embodiment, R6 is

[0284] D

[0285] In one embodiment, D is an active compound; in another embodiment, D is a drug; in another embodiment, D is a cytotoxin; in another embodiment, D is a detection reagent; in another embodiment, D is a diagnostic reagent; in another embodiment, D is a targeting vector.

[0286] In particular, the present invention relates to a linker selected from the group consisting of:

[0287] In particular, the present invention relates to a linker-payload selected from the following table:

[0288] In particular, the present invention relates to an ADC selected from the following table:

[0289] in,

[0290] It represents a monoclonal antibody, preferably an IgG1 and HCAb type monoclonal antibody against HER2 and B7-H3.

[0291] Treatment

[0292] The targeting molecules described herein can be protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. For example, the targeting molecules described herein can be anti-HER2 antibodies, anti-B7-H3 antibodies, including IgG antibodies and HCAb antibodies, or antigen-binding fragments thereof.

[0293] Because the antibody drug conjugates provided herein can target a special cell population, bind to cell surface specific proteins (antigens), and thereby release the drug into the cell in an active form through endocytosis of the conjugate or drug infiltration. Therefore, the antibody drug conjugates of the present application can be used to treat diseases. The antibody drug conjugates of the present application can be administered to a subject (e.g., a human) in a therapeutically effective amount through a suitable route. The subject requiring treatment can be a patient who is at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified by a routine physical examination.

[0294] When treating with the antibody drug conjugate of the present application, delivery can be carried out by the method routine in this area.For example, it can be introduced into the cell by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.Or, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.Other methods can include various transport and carrier systems by using conjugates and biodegradable polymers.

[0295] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health status, the patient's behavior, the patient's diet, the administration time, the administration method, the excretion rate, the combination of drugs, etc.; in addition, the optimal treatment method such as the treatment mode, the daily dosage of the compound described in the application or its pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso-racemate, racemate or tautomer, or a mixture thereof, or the type of pharmaceutically acceptable salt can be verified according to traditional treatment regimens.

[0296] The compounds of the present application may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. When using a pharmaceutical composition, a safe and effective amount of the compounds of the present application may be applied to a mammal (e.g., a human) in need of treatment. The dosage during administration may be a pharmaceutically effective dosage. The specific dosage may also take into account factors such as the route of administration and the patient's health status.

[0297] The present application provides a compound of the present application, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or the use of the pharmaceutical composition of the present application in the preparation of a medicament for treating and / or preventing tumors. For example, the tumor can be selected from tumors associated with the expression of targets such as HER2 and B7-H3. For example, tumors associated with the expression of targets such as HER2 and B7-H3 include tumors with high expression of the target and / or tumors that are positive for the target. For example, the tumor includes solid tumors and / or hematological tumors. For example, the tumor includes breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma or mesothelioma.

[0298] The present application provides a compound, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or a pharmaceutical composition comprising the present application, which can be used to treat and / or prevent tumors. For example, the tumor can be selected from the group consisting of tumors associated with target expression in the following group: HER2, B7-H3, etc. For example, the tumor associated with target expression includes tumors with high expression of the target and / or tumors that are positive for the target. For example, the tumor includes solid tumors and / or hematological tumors. For example, the tumor is selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma.

[0299] The present application provides a method for preventing and / or treating tumors, which may include administering to a subject a compound of the present application, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate, or tautomer thereof, or a pharmaceutical composition of the present application. For example, the tumor may be selected from tumors associated with the expression of the following target: HER2, B7-H3, etc. For example, the tumor associated with the expression of the target includes tumors with high expression of the target and / or tumors positive for the target. For example, the tumor includes solid tumors and / or hematological tumors. For example, the tumor is selected from the following group: breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma.

[0300] Pharmaceutical composition

[0301] The pharmaceutical compositions described herein may contain, in addition to the active compound, one or more excipients, which may be selected from the following groups: fillers (diluents), binders, wetting agents, disintegrants, and excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0302] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as a tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granules, emulsion, hard or soft capsule, or syrup. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and may contain a binder, a filler, a lubricant, a disintegrant, or a pharmaceutically acceptable wetting agent. The composition may also contain one or more ingredients selected from the group consisting of a sweetener, a flavoring agent, a colorant, and a preservative.

[0303] Aqueous suspensions may contain the active ingredient and excipients suitable for preparing aqueous suspensions for mixing. Aqueous suspensions may also contain one or more preservatives, such as one or more colorants, one or more flavorings, and one or more sweeteners. Oil suspensions may be prepared by suspending the active ingredient in a vegetable oil. Oil suspensions may contain a thickening agent. The sweeteners and flavorings mentioned above may also be added.

[0304] The pharmaceutical composition may also be provided as a dispersible powder or granule for preparing an aqueous suspension, wherein the active ingredient is mixed with water to form one or more of a dispersant, a wetting agent, a suspending agent, or a preservative. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. The pharmaceutical composition of the present application may also be in the form of an oil-in-water emulsion.

[0305] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution may then be added to a mixture of water and glycerol to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream via local, bolus injection. Alternatively, the solution or microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous drug delivery device may be used. For example, the device may be an intravenous pump.

[0306] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. Such suspensions may be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents as described herein. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in a parenterally acceptable nontoxic diluent or solvent. Alternatively, a sterile fixed oil may conveniently be used as a solvent or suspending medium.

[0307] The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum, thereby dissolving and releasing the drug in the rectum. Such substances include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

[0308] The pharmaceutical composition of the present application may contain a safe and effective amount of the antibody-drug conjugate of the present application and a pharmaceutically acceptable carrier. Such carriers may include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present application may be prepared in the form of a solution, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical composition may be manufactured under sterile conditions. The dosage of the active ingredient may be a therapeutically effective amount.

[0309] The effective amount of the antibody drug conjugate described in the present application may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors may include, but are not limited to: pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the antibody drug conjugate of the present application is administered at an appropriate dose every day, satisfactory results can be obtained. For example, due to the urgency of the treatment condition, several separate doses can be administered daily, or the dose can be reduced proportionally.

[0310] Example

[0311] The following specific embodiments illustrate the present invention. Those skilled in the art will readily appreciate the advantages and effects of the present invention based on the disclosure herein. Without intending to be bound by any theory, the following examples are intended solely to illustrate the compounds, preparation methods, and uses of the present invention and are not intended to limit the scope of the present invention.

[0312] The definitions of the abbreviations used in this article are as follows:

[0313] ADC (antibody-drug conjugate): antibody-drug conjugate;

[0314] Ala (Alanine): Alanine;

[0315] Boc (t-Butyloxy carbonyl): tert-Butyloxycarbonyl;

[0316] Cit(Citrulline): Citrulline;

[0317] CD276 (Cluster of Differentiation 276): B7-H3;

[0318] DAR (Drug to antibody ratio): antibody drug molar ratio;

[0319] DCC (Dicyclohexylcarbodiimide): Dicyclohexylcarbodiimide;

[0320] DCM (Dichloromethane): dichloromethane;

[0321] DIPEA (N,N-Diisopropylethylamine): N,N-diisopropylethylamine;

[0322] DMAC (Dimethylacetamide): N,N-dimethylacetamide;

[0323] DMF (N,N-Dimethylformamide): N,N-dimethylformamide;

[0324] DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide;

[0325] DX-8951f (Exatecan Mesylate): Exatecan mesylate;

[0326] EA (ethyl acetate): ethyl acetate;

[0327] EEDQ(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline): N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline;

[0328] Fmoc (Fluorenylmethyloxycarbonyl): Fluorenylmethyloxycarbonyl;

[0329] HATU: N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) urea hexafluorophosphate

[0330] HER2 (Human epidermal growth factor receptor 2): human epidermal growth factor receptor 2;

[0331] HIC (Hydrophobic Interaction Chromatography): Hydrophobic interaction chromatography;

[0332] His (Histidine): histidine;

[0333] HOBt (N-Hydroxybenzotrizole): 1-hydroxybenzotriazole;

[0334] m-CPBA (m-Chloroperbenzoic Acid): m-Chloroperbenzoic acid;

[0335] mM (Millimoles): millimole;

[0336] Me(methyl): methyl

[0337] mAB (Monoclonal Antibody): monoclonal antibody;

[0338] MMAE (Monomethyl auristatin E): Monomethyl auristatin E;

[0339] MMAF (Monomethyl auristatin F): Monomethyl auristatin F;

[0340] NHS(N-Hydroxysuccinimide): N-hydroxysuccinimide;

[0341] NAC(N-Acetyl-L-cysteine): N-acetyl-L-cysteine;

[0342] NPC(Bis(4-nitrophenyl)carbonate): Bis(4-nitrophenyl)carbonate

[0343] PAB (p-Aminobenzyl alcohol): p-aminobenzyl alcohol;

[0344] Su / SuOH (Succinimide): succinimide;

[0345] TCEP (Tris(2-carboxyethyl)phosphine): tris(2-carboxyethyl)phosphine;

[0346] TFA (Trifluoroacetic acid): trifluoroacetic acid;

[0347] THF (Tetrahydrofuran): Tetrahydrofuran;

[0348] VA (Valine-Alanine, Val-Ala): valine-alanine dipeptide;

[0349] Val(Valine): valine;

[0350] VC (Valine-Citrulline, Val-Cit): valine-citrulline dipeptide;

[0351] DCU: dicyclohexylurea;

[0352] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0353] TEA: triethylamine;

[0354] Lys(Lysine): lysine;

[0355] Gly (Glycine): glycine;

[0356] Thr (Threonine): threonine;

[0357] Leu (Leucine): Leucine;

[0358] Ile (Isoleucine): Isoleucine;

[0359] Asn(Asparagine): Aspartic acid;

[0360] Phe (Phenylalanine) Phenylalanine.

[0361] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0362] Example 1: Preparation of Compound-1

[0363] The specific synthesis operation is as follows.

[0364] Step 1-1: Preparation of M1

[0365] Fmoc-Ala-NHS (25 g, 57.28 mM) was dissolved in a DME / THF mixed solution (2:1, 300 mL). After all the solution was dissolved, a NaHCO3 solution of citrulline (Cit; 13 g, 74.46 mM) was added and the reaction was stirred at room temperature for 20 hours. LC / MS monitored the complete reaction of compound Fmoc-Ala-NHS, with M1 as the main product. After the reaction was completed, the reaction solution was poured into a 15% aqueous solution of citric acid under stirring to precipitate a jelly-like insoluble material. Filter, drain the filter cake thoroughly, further dry it, and then use ether to beat it twice to obtain product M1 as a white solid powder, 27.88 g, with a yield of about 98%. 1 H NMR (400MHz, DMSO-d6): δ0.89(dd,6H),1.42(m,2H),1.58(m,1H),1.72(m,1H),1.98(m,1H),2.96(q,2H),3.94(t,1H),4.16(q,1H),4.25( m,3H),5.43(s,2H),5.98(t,1H),7.33(t,2H),7.43(q,3H),7.76(t,2H),7.89(d,2H),8.21(d,1H),12.61(s,1H).MS(EI)m / z:497.52[M+H] + ;495.62[MH] - .

[0366] Step 1-2: Preparation of M2

[0367] Compound M1 (15 g, 30.2 mM; 1 eq) was dissolved in a DCM / MeOH mixture (2:1, 450 mL). Once completely dissolved, p-aminobenzyl alcohol (PAB; 7.44 g, 60.4 mM) and EEDQ (14.94 g, 60.4 mM) were added. The mixture was stirred in the dark at room temperature for over 36 hours. After completion, the solvent was removed by concentration under reduced pressure. The resulting residue was slurried three times with diethyl ether to obtain product M2 as a white solid powder (17 g, approximately 95% yield). 1 H NMR (400MHz, DMSO-d6): δ0.86(dd,6H),1.41(m,2H),1.57(m,1H),1.69(m,1H),1.98( m,1H),3.00(m,2H),3.92(q,1H),4.23(q,2H),4.30(q,1H),4.41(q,3H),5.12(t,1H) ,5.42(s,2H),5.98(t,1H),7.23(d,2H),7.32(m,2H),7.41(m,2H),7.46(d,1H),7.54 (d,2H),7.74(t,2H),7.89(d,2H),8.12(d,1H),9.99(s,1H).MS(EI)m / z:602.67[M+H] + ; 624.48[M+Na] + .

[0368] Steps 1-3: Preparation of M3

[0369] Compound M2 (1.5 g, 2.49 mM) was dissolved in DMF (40 mL). After complete dissolution, p-nitrophenol carbonate (NPC; 1.52 g, 4.99 mM) and DIPEA (0.48 g, 3.74 mM) were added. The reaction was stirred at room temperature overnight. After completion, the solvent was removed by concentration under reduced pressure. The resulting residue was slurried three times with diethyl ether. The product M3 was 1.2 g of a white solid powder with a yield of approximately 68%. The resulting material was directly used in the next reaction without further purification. MS (EI) m / z: 767.05 [M+H] + ;789.26[M+Na] + .

[0370] Steps 1-4: Preparation of M4

[0371] Compound M3 (128 mg, 0.167 mM) was dissolved in DMF (40 mL). MMAE (100 mg, 0.139 mM), HOBt (18.82 mg, 0.139), and DIPEA (36 mg, 0.279 mM) were added after complete dissolution. The reaction was stirred at room temperature overnight. After completion, the solvent was removed by concentration under reduced pressure. The resulting residue was slurried three times with ethyl acetate (EA). After filtration, 150 mg of the product M4 was obtained as a white solid powder with a yield of approximately 80%. MS (EI) m / z: 673.80 [M+2H] 2+ .

[0372] Steps 1-5: Preparation of M5

[0373] Compound M4 (135 mg, 0.1 mM) was dissolved in DMF (20 mL), piperidine (1 mL) was added, and the reaction was stirred at room temperature for 2 hours. LC / MS analysis revealed the disappearance of the starting material. The solvent was removed by concentration under reduced pressure, and the resulting residue was slurried with ether and filtered. The filter cake was further purified by column chromatography to afford product M5 as a pale yellow solid powder (83 mg, approximately 74% yield). MS (EI) m / z: 1123.68 [M+H] + ; MS (EI) m / z: 562.41 [M+2H] 2+ .

[0374] Steps 1-6: Preparation of M6

[0375] Methyl 5-chloro-4-nitro-2-picolinate (5 g, 23 mM) and thiourea (5 g, 2.8 eq) were dissolved in sulfolane (30 mL) and heated to 130°C with stirring for 2-4 hours. After completion of the reaction, the reaction solution was allowed to stand and slowly cooled. The product M6 was directly purified by column chromatography to obtain an orange-yellow solid powder (3.68 g), with a yield of approximately 77%. 1 H NMR(400MHz, DMSO-d6)δ8.26(s,2H),7.95(d,J=8.3Hz,1H),7.68(d,J=8.3Hz,1H),3.86(s,3H).MS(EI)m / z:209.29[M+H] + .

[0376] Steps 1-7: Preparation of M7

[0377] M6 (3.77 g, 18 mM) was dispersed in acetonitrile, and copper bromide (8.4 g, 36 mM) was added under stirring at room temperature. After complete dissolution, tert-butyl nitrite (3.71 g, 36 mM) was added dropwise in batches. After the addition was complete, the reaction was stirred at room temperature for about 5 hours. After the reaction was completed, the acetonitrile was removed by concentration under reduced pressure, the residue was redissolved in water, and extracted twice with ethyl acetate. The organic phase was washed twice with water and saturated NaCl solution, respectively, and then dried over Na2SO4 and concentrated to obtain a yellow solid residue. The crude product was further purified by silica gel column chromatography to obtain product M7 as a white solid powder, 4.03 g, with a yield of about 82%. 1 H NMR(400MHz, DMSO-d6)δ8.57(d,J=8.5Hz,1H),8.24(d,J=8.5Hz,1H),3.94(s,3H).MS(EI)m / z:272.74 / 274.84[M+H] + .

[0378] Steps 1-8: Preparation of M8

[0379] M7 (2.05 g, 7.47 mM) was dissolved in tetrahydrofuran (THF, 100 mL). LiOH monohydrate (313 mg, 7.47 mM) was added with stirring at room temperature, followed by approximately 20 mL of water. The reaction was allowed to proceed for approximately 10 minutes. LC / MS monitoring confirmed the complete reaction of compound M7, with compound M8 as the main product. The solvent was removed by concentration under reduced pressure, and the residue was redissolved in water. The pH was adjusted to 3-4 with dilute hydrochloric acid. The suspension was allowed to stand for 15 minutes and then filtered. The filter cake was washed 2-3 times with distilled water and thoroughly dried to obtain product M8 as a light yellow solid powder, 1.8 g, with a yield of approximately 93%. 1 H NMR(400MHz, DMSO-d6)δ13.61(s,1H),8.55(d,J=8.4Hz,1H),8.23(d,J=8.5Hz,1H).MS(EI)m / z:258.83 / 260.73[M+H] + .

[0380] Steps 1-9: Preparation of M9

[0381] M8 (1.80 g, 6.95 mM) was dissolved in a mixture of tetrahydrofuran and MeOH (1:1, 80 mL). An aqueous solution of sodium thiomethoxide (20%, 0.974 g, 13.9 mM) was added with stirring at room temperature. The reaction was stirred at room temperature for approximately 10 minutes. LC / MS analysis confirmed the complete reaction of compound M8, with compound M9 as the primary product. The solvent was removed by concentration under reduced pressure, and the residue was redissolved in water. The pH was first adjusted to ~10 with dilute NaOH solution. After sonication to fully dissolve the residue, the pH was adjusted to 3-4 with dilute hydrochloric acid. The suspension was allowed to stand for 15 minutes and then filtered. The filter cake was washed 2-3 times with distilled water and thoroughly dried to obtain product M9 as a light yellow solid powder, 1.5 g, with a yield of approximately 95%. 1 H NMR (400MHz, DMSO-d6) δ13.26(s,1H),8.31(d,J=8.4Hz,1H),8.16(d,J=8.4Hz,1H),2.85(s,3H).MS(EI)m / z:226.90[M+H] + ;MS(EI)m / z:224.78[MH] - .

[0382] Steps 1-10: Preparation of M10

[0383] M9 (1.3 g, 5.74 mM) was dissolved in DMF (30 mL). Once completely dissolved, NHS (1.32 g, 11.49 mM), DCC (1.78 g, 8.62 mM), and DIPEA (cat., 0.2 mL) were added. The reaction was stirred at room temperature overnight. LC / MS analysis confirmed the complete reaction of compound M9, with compound M10 as the primary product. The insoluble DCU was removed by filtration, and the solvent was removed by concentration under reduced pressure. The resulting residue was directly mixed with crude silica gel and purified by silica gel column chromatography to obtain product M10 as an off-white solid powder (1.78 g, approximately 96% yield). 1 H NMR(400MHz, DMSO-d6)δ8.43(d,J=8.5Hz,1H),8.33(d,J=8.5Hz,1H),2.93(s,4H),2.88(s,3H).MS(EI)m / z:323.98[M+H] + .

[0384] Steps 1-11: Preparation of M11

[0385] M10 (1.0 g, 3.1 mM) was dissolved in DMF (30 mL). After complete dissolution, 6-aminohexanoic acid (0.61 g, 4.65 mM) and a catalytic amount of DIPEA were added. The reaction was stirred at room temperature overnight. After completion, the solvent was removed by concentration under reduced pressure to obtain a pale yellow solid residue. The product M11 was purified by silica gel column chromatography as a white solid powder (916 mg, approximately 87% yield). 1 H NMR (500MHz, DMSO-d6): δ12.01(br,1H),8.16(d,J=7.7Hz,1H),8.06(d,J=7.5Hz,1H),8.01(t,1H),3.35(q,J=5.4Hz,2H ),2.77(s,3H),2.26(t,J=8.9Hz,2H),1.62-1.56(m,2H),1.59-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m / z:340.06[M+H] + ;MS(EI)m / z:338.08[MH] - .

[0386] Steps 1-12: Preparation of M12

[0387] M11 (500 mg, 1.47 mM) was dissolved in DCM (30 mL). After complete dissolution, m-chloroperbenzoic acid (m-CPBA, 85%; 1.0 g, 5.89 mM) was added. The reaction was stirred at room temperature overnight. LC / MS analysis confirmed the complete reaction of compound M11, with compound M12 as the primary product. The solvent was removed by concentration under reduced pressure. The resulting residue was mixed with crude silica gel and purified by silica gel column chromatography to obtain product M12 as a white solid powder (509 mg, approximately 93% yield). 1 H NMR (500MHz, DMSO-d6) δ8.21(d,J=7.5Hz,1H),8.09(d,J=7.5Hz,1H),8.01(t,1H),3.48(s,3H),3.35(q,J=5.4H z,2H),2.26(t,J=8.9Hz,2H),1.61-1.55(m,2H),1.58-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m / z:372.08[M+H] + ;MS(EI)m / z:370.05[MH] - .

[0388] Step 1-13: Preparation of Compound-1

[0389] M5 (50 mg, 0.0445 mM) and M12 (20 mg, 0.0534 mM) were dissolved in DMF (10 mL), stirred until completely dissolved, and the reaction system was transferred to an ice bath for cooling. HATU (25.4 mg, 0.668 mM) and a catalytic amount of DIPEA were then added to the reaction solution. After the addition was completed, the reaction was stirred in an ice bath for 30 minutes, then gradually returned to room temperature and stirred at room temperature for overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the resulting residue was directly mixed with crude silica gel and purified by silica gel column chromatography to obtain compound-1 as a white solid, which was further lyophilized to a fluffy white solid of 36.8 mg, with a yield of approximately 56%. MS (EI) m / z: 738.38 [M+2H] 2+ ; MS (ESI) m / z: 1476.7643 [M+H] + .

[0390] Example 2: Preparation of Compound-2

[0391] The specific synthesis operation is as follows.

[0392] Step 2-1: Preparation of M13

[0393] Following the synthesis method of M4, Dx-8951 mesylate was used to replace MMAE and react with M3 to obtain M13, a pale yellow solid powder with a yield of approximately 83%. MS (EI) m / z: 1063.45 [M+H] + .

[0394] Step 2-2: Preparation of M14

[0395] Following the synthesis method of M5, M13 was used to replace M4 and the protecting group was removed in a piperidine solution to obtain M14 as a pale yellow solid powder in a yield of approximately 70%. MS (EI) m / z: 841.41 [M+H] + .

[0396] Step 2-3: Preparation of Compound-2

[0397] Following the synthesis method of compound-1, M14 was used to replace M5 and react with M12 to obtain compound-2, a light yellow solid powder, with a yield of approximately 56%. MS (EI) m / z: 597.68 [M+2H] 2+ ; MS (ESI) m / z: 1194.4175 [M+H] + .

[0398] Example 3: Preparation of Compound-3

[0399] The specific synthesis operation is as follows.

[0400] Step 3-1: Preparation of M15

[0401] 1-Amino-3,6,9,12-tetraoxapentadecan-15-oic acid was used to replace 6-aminohexanoic acid to react with M10. M15 was prepared according to the synthesis method of M11 as a light yellow viscous oil with a yield of about 92%. 1 H NMR(500MHz,Chloroform-d)δ12.04(br,1H),8.26(t,1H),8.16(d,J=7.7Hz,1H),8.06(d,J=7.5Hz,1H),3.69-3.59( m,15H),3.59-3.53(m,2H),3.40(dt,J=5.6,4.2Hz,2H),2.77(s,3H),2.48(t,J=6.4Hz,2H).MS(EI)m / z:474.07[M+H] + .

[0402] Step 3-2: Preparation of M16

[0403] According to the synthesis method of M12, M15 was used to replace M11 and react with m-CPBA to prepare M16, a light yellow viscous oil, with a yield of about 80%. 1 H NMR(500MHz,Chloroform-d)δ12.04(br,1H),8.26(t,1H),8.16(d,J=7.7Hz,1H),8.06(d,J=7.5Hz,1H),3.69-3.59( m,15H),3.59-3.53(m,2H),3.40(dt,J=5.6,4.2Hz,2H),2.77(s,3H),2.48(t,J=6.4Hz,2H).MS(EI)m / z:505.98[M+H] + ;528.10[M+Na] + .

[0404] Step 3-3: Preparation of Compound-3

[0405] According to the synthesis method of compound-1, M16 was used to replace M12 and react with M5 to prepare compound-3, a white solid powder with a yield of about 63%. MS (EI) m / z: 805.92 [M+2H] 2+ ; MS (ESI) m / z: 1610.8215 [M+H] + .

[0406] Example 4: Preparation of Compound-4

[0407] The specific synthesis operation is as follows.

[0408] Step 4-1: Preparation of Compound-4

[0409] Following the synthesis method of compound-2, M16 was used to replace M12 and react with M14 to obtain compound-4, a pale yellow solid powder, with a yield of approximately 55%. MS (EI) m / z: 664.71 [M+2H] 2+ ; MS (ESI) m / z: 1328.4768 [M+H] + .

[0410] Example 5: Preparation of Compound-5

[0411] The specific synthesis operation is as follows.

[0412] Step 5-1: Preparation of M17

[0413] According to the synthesis method of M1, L-alanine was used to replace L-citrulline and react with Fmoc-Ala-NHS to prepare compound M17 as a white solid powder with a yield of about 83%. 1 H NMR (400MHz, DMSO-d6): δ0.88(dd,6H),1.27(d,3H),1.96(m,1H),3.89(q,1H),4.22(m,4H),7.33(t,2H),7.43(m,3H),7.74(t 2H),7.89(d,2H),8.25(d,1H),12.48(s,1H).MS(EI)m / z:411.23[M+H] + ;455.05[M+Na] + .

[0414] Step 5-2: Preparation of M18

[0415] According to the synthesis method of M2, M1 was replaced by M17, and EEDQ and PAB were subjected to condensation reaction to prepare compound M18 as a white solid powder with a yield of about 87%. 1H NMR (400MHz, DMSO-d6): δ0.88(dd,6H),1.30(d,3H),2.00(m,1H),3.91(t,1H),4.22(q,2H),4.30(t,1H),4.40(br,1H),4.42(d,2H),5.13 (t,1H),7.24(t,2H),7.34(t,2H),7.41(t,2H),7.52(q,3H),7.75(t,2H),7.89(d,2H),8.22(d,1H),9.96(s,1H).MS(EI)m / z:516.29[M+H] + ;538.20[M+Na] + .

[0416] Step 5-3: Preparation of M19

[0417] Following the synthesis of M3, M18 was substituted for M2 and reacted with p-nitrophenol carbonate to produce compound M19, a white solid powder in approximately 92% yield. The resulting material was directly used in the next reaction without further purification. MS (EI) m / z: 681.24 [M+H] + ;703.20[M+Na] + .

[0418] Step 5-4: Preparation of M20

[0419] Following the synthesis method of M4, M19 was used to replace M3 and react with MMAE to obtain compound M20, a pale yellow solid powder, in a yield of approximately 67%. MS (EI) m / z: 630.34 [M+2H] 2+ ; MS (ESI) m / z: 1259.7341 [M+H] + .

[0420] Step 5-5: Preparation of M21

[0421] Following the synthesis method for M5, M20 was used to replace M4 and the Fmoc protecting group was removed in piperidine to obtain compound M21 as a pale yellow solid powder in approximately 55% yield. The product was somewhat hygroscopic and was not readily allowed to proceed directly to the next reaction. MS (EI) m / z: 1123.68 [M+H] + .

[0422] Step 5-6: Preparation of Compound-5

[0423] Following the synthesis method of compound-1, M21 was used to replace M5 and react with compound M12 to obtain compound-5 as an off-white solid powder with a yield of approximately 53%. MS (EI) m / z: 695.86 [M+2H] 2+ ; MS (ESI) m / z: 1390.7162 [M+H] + .

[0424] Example 6: Preparation of Compound-6

[0425] The specific synthesis operation is as follows.

[0426] Step 6-1: Preparation of Compound-6

[0427] Following the synthesis method of compound-5, M16 was used to replace M12 and M21 to react with the compound to obtain compound-6, an off-white solid powder, with a yield of approximately 61%. MS (EI) m / z: 762.93 [M+2H] 2+ ; MS (ESI) m / z: 1524.7745 [M+H] + .

[0428] Example 7: Preparation of Compound-7

[0429] The specific synthesis operation is as follows.

[0430] Step 7-1: Preparation of M22

[0431] Following the synthesis method of M13, M19 was used to replace M3 and condensed with Dx-8951f to obtain M22 as a pale yellow solid powder in approximately 85% yield. MS (EI) m / z: 977.40 [M+H] + .

[0432] Step 7-2: Preparation of M23

[0433] Following the synthesis method of M14, M22 was used to replace M13 and the Fmoc protecting group was removed in a piperidine solution to obtain M23 as a pale yellow solid powder in approximately 75% yield. MS (EI) m / z: 755.36 [M+H] + .

[0434] Step 7-3: Preparation of Compound-7

[0435] Following the synthesis method of compound 2, M23 was substituted for M14 and condensed with M12 under the action of HATU to obtain compound 7 as a pale yellow solid powder with a yield of approximately 63%. MS (EI) m / z: 1108.33 [M+H]+ ; MS (ESI) m / z: 1130.32 [M+Na] + .

[0436] Example 8: Preparation of Compound-8

[0437] The specific synthesis operation is as follows.

[0438] Step 8-1: Preparation of Compound-8

[0439] Following the synthesis method of compound 7, M16 was used to replace M12 and condensed with M23 under the action of HATU to obtain compound 8 as a pale yellow solid powder with a yield of approximately 56%. MS (EI) m / z: 621.74 [M+2H] 2+ ; MS (ESI) m / z: 1242.4294 [M+H] + .

[0440] Example 9: Preparation of Compound-9

[0441] The specific synthesis operation is as follows.

[0442] Step 9-1: Preparation of M24

[0443] The benzyloxycarbonyl-protected diglycine (10 g, 37.56 mM) was dissolved in acetonitrile (100 mL), and NHS (4.75 g, 41.31 mM) and EDCI (8.64 g, 45.07 mM) were added under stirring. Under nitrogen protection, the mixture was stirred and reacted overnight. After the reaction was completed, the solvent was removed under reduced pressure and concentrated to obtain a colorless oily residue. The oil was redissolved in ethyl acetate, washed with distilled water and separated, and repeated 3 times. The resulting organic phase was washed 3 times with saturated brine, and the reaction solution was concentrated to about 100 mL. After standing at room temperature for 24 hours, white insoluble matter precipitated, filtered and the filter cake was washed with EA. After the filter cake was fully dried, the product M24 was obtained as a white solid powder, 13 g, with a yield of about 96%. M24 is an active ester intermediate and is directly subjected to the next step of the reaction without long-term storage. MS (EI) m / z: 364.18 [M+H] + ; MS (EI) m / z: 386.10 [M+Na] + .

[0444] Step 9-2: Preparation of M25

[0445] Phenylalanine (4.55 g, 27.52 mM) was dissolved in a mixed solution of acetonitrile and water (1:1; 100 mL), triethylamine (TEA, 2.78 g, 27.52 mM) was added and stirred at room temperature for 5 minutes to completely dissolve the solid. M24 (10 g, 27.52 mM) was added and the reaction was continued with stirring at room temperature for 3 hours. After the reaction was completed, water (50 mL) was added to the reaction solution, and hydrochloric acid was added dropwise while stirring to adjust the pH to 2. Most of the acetonitrile was removed by concentration under reduced pressure, and the remaining liquid was left at room temperature overnight to precipitate a white solid. The filter cake was filtered and washed with ether. After sufficient drying, the product M25 was obtained as a white solid powder, 13.2 g, with a yield of approximately 84%. 1 H NMR (400MHz, DMSO-d6) δ7.77(d,J=9.1Hz,1H),7.48(t,J=6.0Hz,1H),7.38-7.18(m,10H),5.89(t,J=6.6Hz,1H),5.10(d,J=0 .8Hz,2H),4.45(dt,J=9.2,7.0Hz,1H),3.86(d,J=6.6Hz,2H),3.84-3.71(m,2H),3.12-3.06(m,2H).MS(EI)m / z:414.19[M+H] + ;MS(EI)m / z:412.13[MH] - .

[0446] Step 9-3: Preparation of M26

[0447] Fmoc-protected diglycine (12.49 g, 35.24 mM) was dispersed in a mixed solution of tetrahydrofuran (200 mL) and acetic acid (50 mL), heated to 40 ° C and all solids dissolved under stirring. After adding lead tetraacetate (25 g, 56.39 mM), the temperature was raised to reflux for 2 hours. After the reaction was completed, the heating was stopped and the mixture was cooled naturally. Under stirring, about 100 mL of 20% sodium citrate solution was added to the mixture to separate out a light yellow precipitate. After filtering to remove insoluble matter, the filtrate was washed 2 to 3 times with 20% sodium citrate solution. The organic phase obtained by separation was concentrated to about 60 mL, and about 100 mL of distilled water was added to the concentrated solution. After standing overnight at room temperature, it was filtered. The filter cake was washed 3 times with distilled water and fully drained to obtain M26 as a white solid powder, 13.2 g, with a yield of about 99%.

[0448] 1H NMR(400MHz,Chloroform-d)δ7.82(dd,J=8.2,1.3Hz,2H),7.62(dd,J=7.6,1.4Hz,2H),7.53-7.46(m,3H),7.41(td,J=7.8,1.5Hz,2H),5.81( t,J=6.6Hz,1H),5.44-5.39(m,1H),5.04(d,J=3.5Hz,2H),4.42(d,J=4.8Hz,2H),3.84(d,J=6.6Hz,2H),2.19(s,3H).MS(EI)m / z:369.21[M+H] + .

[0449] Step 9-4: Preparation of M27

[0450] M26 (12.2 g, 33.12 mM) was dissolved in ethylene glycol dimethyl ether (DME; 200 mL), and benzyl glycolate (11 g, 66.23 mM) was added to the reaction mixture. After the solid was completely dissolved, the reaction solution was placed in an ice bath for cooling. 10 M NaOH (10 eq, 1.32 g) solution was added dropwise to the reaction solution, and the reaction was stirred at low temperature for 1 hour. About 1.3 mL of glacial acetic acid was added dropwise to the reaction solution, and the mixture was continued to react at low temperature for 1 hour. After the reaction was completed, 150 mL of water was added to the reaction system, and stirring was continued at low temperature for 2.5 hours. Filter, and the filter cake was washed three times with a mixed solution of water and ethylene glycol dimethyl ether (1:1) and fully dried to obtain M27 as a white solid powder, 13.94 g, with a yield of about 89%. The obtained solid product was directly used in the next reaction. MS (EI) m / z: 475.22 [M+H] + .

[0451] Step 9-5: Preparation of M28

[0452] M27 (13 g, 23.4 mM) was dissolved in acetonitrile (400 mL), and DBU (4.59 g, 30.14 mM) was added to the reaction mixture. The mixture was stirred and reacted at room temperature for 4 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a viscous oily residue. The residue was further purified by silica gel column chromatography with a yield of >100%, but it was suspected that the residue contained unremoved solvent. The resulting free amine product M28 had poor stability and was used directly in the next reaction. MS (EI) m / z: 253.23 [M+H] + .

[0453] Step 9-6: Preparation of M29

[0454] M25 (9.0 g, 22 mM) and M28 (6.3 g, 25 mM) were dissolved in acetonitrile (200 mL). After adding a catalytic amount of DIPEA (0.5 eq), the reaction system was placed in an ice bath and cooled to about 0 ° C. EDCI (5.75 g, 30 mM) was added in batches. The reaction was continued at low temperature for 1 hour and then transferred to room temperature for 2 hours. After the reaction was completed, the solvent was removed under reduced pressure and concentrated to obtain a yellow oily residue. It was redissolved in ethyl acetate, washed with water and separated 3 times, and the organic solvent was concentrated to about 100 mL. Stirred at room temperature overnight, the precipitated paste-like insoluble matter was filtered, fully drained and fully dried to obtain M29 as a light yellow solid powder, 13.4 g, with a yield of about 94%. MS (EI) m / z: 648.33 [M+H] + ; MS (EI) m / z: 670.26 [M+Na] + .

[0455] Step 9-7: Preparation of M30

[0456] M29 (1.0 g, 1.54 mM) was dissolved in methanol (50 mL). LiOH monohydrate (77.7 mg, 1.85 mM) and a catalytic amount of water were added with stirring at room temperature. The reaction was allowed to proceed overnight at room temperature. The solvent was then concentrated under reduced pressure to remove the solvent. The resulting solid residue was mixed with crude silica gel and purified by silica gel column chromatography to afford product M30 as a white solid powder (300 mg, approximately 35% yield). MS (EI) m / z: 580.31 [M+Na] + ;MS(EI)m / z:556.24[MH] - .

[0457] Step 9-8: Preparation of M31

[0458] M30 (100 mg, 0.179 mM) was dissolved in DMF (20 mL). After the solid was completely dissolved, Dx-8951f methanesulfonate (64 mg, 0.1196 mM), HATU (136.39 mg, 0.359 mM), and DIPEA (92.73 mg, 0.7174 mM) were added in sequence. After stirring at room temperature for 4 hours, the solvent was removed by concentration under reduced pressure. The resulting oily residue was purified by silica gel column chromatography to obtain product M31 (210 mg, yield >100%), suspected to be coated with a small amount of silica gel. The crude product was directly carried to the next step without further purification. MS (EI) m / z: 975.41 [M+H] + .

[0459] Step 9-9: Preparation of M32

[0460] Following the synthesis of M14, M31 was substituted for M13 and the Fmoc protecting group was removed in piperidine to produce M32 as a pale yellow solid powder in approximately 57% yield. The resulting amino product was unstable and was used directly in the next reaction without further purification. MS (EI) m / z: 841.45 [M+H] + .

[0461] Steps 9-10: Preparation of Compound-9

[0462] Following the synthesis method of compound-2, M32 was substituted for M14 and condensed with M12 under the action of HATU to obtain compound-9 as a pale yellow solid powder with a yield of approximately 45%. MS (EI) m / z: 597.72 [M+2H] 2+ ; MS (ESI) m / z: 1194.3832 [M+H] + .

[0463] Example 10: Preparation of Compound-10

[0464] The specific synthesis operation is as follows.

[0465] Step 10-1: Preparation of Compound-10

[0466] Following the synthesis method of compound-9, M16 was used to replace M12 and condensed with M32 under the action of HATU to obtain compound-10 as a pale yellow solid powder with a yield of approximately 47%. MS (EI) m / z: 664.69 [M+2H] 2+ ; MS (ESI) m / z: 1328.4436 [M+H] + .

[0467] Example 11: Preparation of Compound-11

[0468] The specific synthesis operation is as follows.

[0469] Step 11-1: Preparation of M33

[0470] Following the synthesis of compound M31, MMAE was used to replace the mesylate salt of Dx-8951f. The mixture reacted with M30 in the presence of HATU to produce the intermediate product M33, a white solid powder, in approximately 78% yield. MS (EI) m / z: 629.33 [M+2H]. 2+ ; MS (ESI) m / z: 1257.7135 [M+H] + .

[0471] Step 11-2: Preparation of M34

[0472] Compound M33 (100 mg, 0.08 mM) was dissolved in methanol (10 mL), and a catalytic amount of Pd / C (~15 mg) was added. The mixture was stirred under a hydrogen atmosphere. After 3 hours of reaction at room temperature, the hydrogen balloon was removed, the insoluble matter was filtered out, and the mixture was concentrated under reduced pressure to obtain a pale yellow glassy residue. Compound M34 was further purified by silica gel column chromatography to obtain a white solid powder (61 mg, approximately 67% yield). MS (ESI) m / z: 1123.68 [M+H] + .

[0473] Step 11-3: Preparation of Compound-11

[0474] Following the synthesis method of compound 1, M34 was used to replace M5 and condensed with M12 under the action of HATU to obtain compound 11 as a white solid powder with a yield of approximately 46%. MS (EI) m / z: 738.79 [M+2H] 2+ ; MS (ESI) m / z: 1476.7281 [M+H] + .

[0475] Example 12: Preparation of Compound-12

[0476] The specific synthesis operation is as follows.

[0477] Step 12-1: Preparation of Compound-12

[0478] According to the synthesis method of compound-11, M16 was used to replace M12 and condensed with M34 under the action of HATU to prepare compound-12 as a white solid powder with a yield of about 53%. MS (EI) m / z: 805.95 [M+2H] 2+ ; MS (ESI) m / z: 1610.7848 [M+H] + .

[0479] Example 13: Preparation of Compound-13

[0480] The specific synthesis operation is as follows.

[0481] Step 13-1: Preparation of M35

[0482] M2 (1.0 g, 1.66 mM) was dissolved in DMF (15 mL). After the solid was completely dissolved, piperidine (1 mL) was added and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting oily residue was purified by silica gel column chromatography to obtain a colorless viscous liquid. The product was further dried under reduced pressure and allowed to stand to obtain M35 as a white solid, 530 mg, with a yield of approximately 84%. 1 H NMR (400MHz, DMSO-d6): δ0.86(dd,6H),1.38(m,2H),1.64(m,2H),1.94(m,1H),2.95(m,2H),3.05(d,1H),4.43(s,2H),4.47(s, 1H),5.13(br,1H),5.44(d,2H),6.01(br,1H),7.23(d,2H),7.54(d,2H),8.17(br,1H),10.07(s,1H).MS(EI)m / z:380.32[M+H] + ; MS (EI) m / z: 402.31 [M+Na] + .

[0483] Step 13-2: Preparation of M36

[0484] 2,5,8,11-Tetrahydrotetradecane-14-oic acid (5.0 g, 21.16 mM) was dissolved in dichloromethane (50 mL), and NHS (3.65 mg, 31.74 mM) and EDCI (4.78 g, 25.4 mM) were added sequentially. The reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting colorless oil residue was further purified by column chromatography to obtain M36, a colorless viscous oil, 8.0 g, with a yield of >100%. The oil contained a small amount of solvent that was not evaporated. The obtained oil was used directly in the next reaction as soon as possible without further purification. MS (EI) m / z: 334.42 [M+H] + .

[0485] Step 13-3: Preparation of M37

[0486] Fmoc- 2 N-Boc- 6NL-Lys (10.0 g, 21.3 mM) was dispersed in DCM (100 mL) but was incompletely dissolved. Addition of TFA (10 mL) completely dissolved the reactant, resulting in a light yellow color. After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to remove the solvent, redissolved in ethyl acetate, and re-evaporated to dryness. This process was repeated three times to remove any residual TFA. The mixture was then fully pumped to dryness using a diaphragm pump, yielding 17 g of a viscous oil. The yield was >100%, although a small amount of solvent was still present. The resulting colorless oil, M37, was used directly in the next step. MS (EI) m / z: 369.21 [M+H] + .

[0487] Step 13-4: Preparation of M38

[0488] Compounds M37 (crude, ~15 mM) and M36 (crude, ~15 mM) were dispersed in DCM (100 mL), and DIPEA (1.94 g, 15 mM) was added. The mixture was stirred and reacted at room temperature overnight. The mixture was concentrated under reduced pressure and thoroughly pumped to dryness using a diaphragm pump to obtain a crude oil. Further purification by silica gel column chromatography afforded product M38 as a colorless, viscous oil (13 g, yield >100%), containing a small amount of unremoved solvent. The resulting colorless oil, M38, was used directly in the next reaction. MS (EI) m / z: 587.32 [M+H] + .

[0489] Step 13-5: Preparation of M39

[0490] Compounds M38 (~2.0 g, 3.4 mM) and M35 (1.0 g, 2.6 mM) were dissolved in DMF (20 mL) and stirred at room temperature until completely dissolved. HATU (1.50 g, 3.93 mM) and DIPEA (0.508 g, 3.93 mM) were then added sequentially. The reaction was stirred at room temperature overnight and concentrated under reduced pressure to remove the solvent, yielding a glassy residue. Ethyl acetate was added to the residue and ultrasonically dispersed and slurried. The paste was filtered and the filter cake was washed with ethyl acetate (repeated 3 times). The filter cake was thoroughly dried to yield M39 as a light yellow solid powder, 2.5 g, with a yield of approximately 95%. MS (EI) m / z: 948.56 [M+H] + ; MS (EI) m / z: 983.17 [M+Cl] - .

[0491] Step 13-6: Preparation of M40

[0492] Following the synthesis of compound M3, M39 was used in place of M2 to react with p-nitrophenol carbonate to obtain intermediate product M40, a white solid powder, in approximately 74% yield. MS (EI) m / z: 1113.69 [M+H] + ; MS (ESI) m / z: 1135.49 [M+Na]+ .

[0493] Step 13-7: Preparation of M41

[0494] Following the synthesis of compound M13, M40 was substituted for M3 and reacted with Dx-8951f mesylate to obtain intermediate M41, a pale yellow solid powder, in approximately 57% yield. MS (EI) m / z: 705.27 [M+2H] 2+ ; MS (ESI) m / z: 1409.6475 [M+H] + .

[0495] Step 13-8: Preparation of M42

[0496] Following the synthesis of compound M14, M41 was used to replace M13 and the Fmoc protecting group was removed in a piperidine solution to obtain the intermediate product M42 as a dusty solid powder in approximately 68% yield. MS (EI) m / z: 594.37 [M+2H] 2+ ; MS (ESI) m / z: 1187.5790 [M+H] + .

[0497] Step 13-9: Preparation of Compound-13

[0498] Following the synthesis method of compound-2, M42 was substituted for M14 and condensed with M12 under the action of HATU to obtain compound-13 as a white solid powder with a yield of approximately 59%. MS (EI) m / z: 770.86 [M+2H] 2+ ; MS (ESI) m / z: 1540.6295 [M+H] + .

[0499] Example 14: Preparation of Compound-14

[0500] The specific synthesis operation is as follows.

[0501] Step 14-1: Preparation of Compound-14

[0502] Following the synthesis method of compound 13, M16 was used to replace M12 and condensed with M42 under the action of HATU to obtain compound 14 as a white solid powder with a yield of approximately 43%. MS (EI) m / z: 838.13 [M+2H] 2+ ; MS (ESI) m / z: 1674.6877 [M+H] + .

[0503] Example 15: Preparation of Compound-15

[0504] The specific synthesis operation is as follows.

[0505] Step 15-1: Preparation of M43

[0506] Following the synthesis method of compound M4, M40 was used to replace M3 and react with MMAE to obtain M43, a white solid powder, in a yield of approximately 76%. MS (EI) m / z: 846.57 [M+2H] 2+ ; MS (ESI) m / z: 1691.9921 [M+H] + .

[0507] Step 15-2: Preparation of M44

[0508] Following the synthesis of compound M5, M43 was used to replace M4 and the Fmoc protecting group was removed in a piperidine solution to obtain M44, a glassy solid powder, in a yield of approximately 61%. MS (EI) m / z: 735.54 [M+2H] 2+ ; MS (ESI) m / z: 1469.9233 [M+H] + .

[0509] Step 15-3: Preparation of Compound-15

[0510] Following the synthesis method of compound 1, M44 was used to replace M5 and condensed with M12 under the action of HATU to obtain compound 15 as a white solid powder with a yield of approximately 48%. MS (EI) m / z: 911.95 [M+2H] 2+ ; MS (ESI) m / z: 1822.9741 [M+H] + .

[0511] Example 16: Preparation of Compound-16

[0512] The specific synthesis operation is as follows.

[0513] Step 16-1: Preparation of Compound-16

[0514] Following the synthesis method of compound 15, M16 was used to replace M12 and condensed with M44 under the action of HATU to obtain compound 16 as a white solid powder with a yield of approximately 59%. MS (EI) m / z: 979.23 [M+2H] 2+ ; MS (ESI) m / z: 1957.0324 [M+H] + .

[0515] Example 17: Preparation of Compound-17

[0516] The specific synthesis operation is as follows.

[0517] Step 17-1: Preparation of M45

[0518] Following the synthesis of M35, M18 was substituted for M2, and the Fmoc protecting group was removed in piperidine to obtain M45, a colorless, viscous oil in approximately 75% yield. M45 is unstable at room temperature and should be used in the next reaction as soon as possible. 1 H NMR (400MHz, DMSO-d6): δ0.85(dd,6H),1.29(d,3H),1.92(m,1H),2.80(d,1H),3.00(d,1H),4.43(s,1H) ,4.48(t,1H),5.13(s,1H),7.24(d,2H),7.53(d,2H),8.18(s,1H),10.0(s,1H).MS(EI)m / z:294.23[M+H] + ; MS (EI) m / z: 316.22 [M+Na] + .

[0519] Step 17-2: Preparation of M46

[0520] Following the synthesis method of M39, M45 was used to replace M35 and acylated with M38 in the presence of HATU to produce M46, a pale yellow solid powder, in approximately 68% yield. MS (EI) m / z: 862.48 [M+H] + ; MS (EI) m / z: 884.46 [M + Na] + .

[0521] Step 17-3: Preparation of M47

[0522] Following the synthesis method of M40, M46 was used to replace M39 and reacted with p-nitrophenol carbonate to obtain M47, a pale yellow solid powder, in approximately 85% yield. MS (EI) m / z: 1027.47 [M+H] + ; MS (EI) m / z: 1049.45 [M+Na] + .

[0523] Step 17-4: Preparation of M48

[0524] Following the synthesis of M41, M47 was used to replace M40 and reacted with Dx-8951f mesylate to obtain M48, an off-white solid powder, in approximately 44% yield. MS (EI) m / z: 662.32 [M+2H] 2+; MS (ESI) m / z: 1323.5974 [M+H] + .

[0525] Step 17-5: Preparation of M49

[0526] Following the synthesis of M42, M48 was used to replace M41 and the Fmoc protecting group was removed in a piperidine-DMF solution to obtain M49 as a dusty gray solid powder in approximately 56% yield. MS (EI) m / z: 1101.55 [M+H] + ; MS (EI) m / z: 1123.54 [M+Na] + .

[0527] Step 17-6: Preparation of Compound-17

[0528] Following the synthesis of compound 13, M49 was substituted for M42 and acylated with M12 under the action of HATU to yield compound 17 as an off-white solid powder in approximately 49% yield. MS (EI) m / z: 727.72 [M+2H] 2+ ; MS (ESI) m / z: 1454.5831 [M+H] + .

[0529] Example 18: Preparation of Compound-18

[0530] The specific synthesis operation is as follows.

[0531] Step 18-1: Preparation of Compound-18

[0532] Following the synthesis of compound 17, M16 was substituted for M12 and acylated with M49 in the presence of HATU to yield compound 18 as an off-white solid powder in approximately 50% yield. MS (EI) m / z: 794.76 [M+2H] 2+ ; MS (ESI) m / z: 1588.6387 [M+H] + .

[0533] Example 19: Preparation of Compound-19

[0534] The specific synthesis operation is as follows.

[0535] Step 19-1: Preparation of M50

[0536] Following the synthesis of M48, MMAE was used to replace the mesylate salt of Dx-8951f to react with M47 for acylation to produce M50, a white solid powder, in approximately 61% yield. MS (EI) m / z: 803.49 [M+2H] 2+ .

[0537] Step 19-2: Preparation of M51

[0538] Following the synthesis of M5, M50 was used to replace M4 and the Fmoc protecting group was removed in a piperidine-DMF solution to obtain M51, a white waxy solid in approximately 68% yield. MS (EI) m / z: 692.43 [M+2H] 2+ ; MS (ESI) m / z: 1383.8751 [M+H] + .

[0539] Step 19-3: Preparation of Compound-19

[0540] Following the synthesis of compound 1, M51 was substituted for M5 and acylated with M12 under the action of HATU to yield compound 19 as a white solid powder in approximately 57% yield. MS (EI) m / z: 868.89 [M+2H] 2+ ; MS (ESI) m / z: 1736.9256 [M+H] + .

[0541] Example 20: Preparation of Compound-20

[0542] The specific synthesis operation is as follows.

[0543] Step 20-1: Preparation of Compound-20

[0544] Following the synthesis of compound 19, M16 was substituted for M12 and acylated with M51 under the action of HATU to yield compound 20 as a white solid powder in approximately 60% yield. MS (EI) m / z: 936.02 [M+2H] 2+ ; MS (ESI) m / z: 1870.9833 [M+H] + .

[0545] Example 21: Preparation of Compound-21

[0546] The specific synthesis operation is as follows.

[0547] Step 21-1: Preparation of M52

[0548] According to the synthesis method of M1, Boc-6 NL-Lys replaced citrulline and reacted with Fmoc-Val-NHS to produce M52 as a white solid powder. The yield was approximately 96%. MS (EI) m / z: 568.35 [M+H] + ; MS (EI) m / z: 590.28 [M+Na] + .

[0549] Step 21-2: Preparation of M53

[0550] Following the synthesis method of M2, M52 was substituted for M1 and condensed with PAB in the presence of EEDQ to produce M53, a white solid powder. The yield was approximately 89%. MS (EI) m / z: 673.33 [M+H] + ; MS (EI) m / z: 695.31 [M+Na] + .

[0551] Step 21-3: Preparation of M54

[0552] Following the synthesis of M3, M53 was substituted for M2 in the presence of p-nitrophenol carbonate to produce M54 as a white solid powder. The yield was approximately 91%. MS (EI) m / z: 838.35 [M+H] + .

[0553] Step 21-4: Preparation of M55

[0554] Following the synthesis of M13, M54 was substituted for M3 in an acylation reaction with Dx-8951f to produce M55, a gray solid powder. The yield was approximately 49%. MS (EI) m / z: 1134.52 [M+H] + ; MS (EI) m / z: 1156.51 [M+Na] + .

[0555] Step 21-5: Preparation of M56

[0556] Following the synthesis of M14, M55 was used to replace M13 and the Fmoc protecting group was removed in a piperidine-DMF solution to obtain M56, an off-white glassy solid. The yield was approximately 55%. MS (EI) m / z: 912.43 [M+H] + ; MS (EI) m / z: 934.41 [M+Na] + .

[0557] Step 21-6: Preparation of M57

[0558] Following the synthesis of Compound 2, M56 was substituted for M14 and acylated with M12 under the action of HATU to produce M57, an off-white solid powder, in a yield of approximately 46%. MS (EI) m / z: 633.28 [M+2H] 2+ ; MS (ESI) m / z: 1265.4821 [M+H] + .

[0559] Step 21-7: Preparation of Compound-21

[0560] Compound M57 (50 mg, 0.079 mM) was dispersed in DCM (10 mL) and HCOOH (1 mL) was added with stirring to dissolve the entire M57. After stirring at room temperature for 4 hours, the solvent was removed by concentration under reduced pressure to yield a pale yellow semisolid oily residue. This residue was redissolved in crude silica gel and further purified by silica gel column chromatography to yield compound 21 as an off-white glassy solid (47 mg, approximately 51% yield). MS (EI) m / z: 583.23 [M+2H]. 2+ ; MS (ESI) m / z: 1165.4287 [M+H] + .

[0561] Example 22: Preparation of Compound-22

[0562] The specific synthesis operation is as follows.

[0563] Step 22-1: Preparation of M58

[0564] Following the synthesis of M57, M16 was substituted for M12 and acylated with M56 in the presence of HATU to yield M58, an off-white glassy solid, in approximately 49% yield. MS (EI) m / z: 700.32 [M+2H] 2+ ; MS (ESI) m / z: 1399.5392 [M+H] + .

[0565] Step 22-2: Preparation of Compound-22

[0566] Following the synthesis of compound 21, M58 was used to replace M57 and the Boc protecting group was removed in formic acid solution to obtain compound 22, a gray glassy solid, in a yield of approximately 52%. MS (EI) m / z: 650.29 [M+2H] 2+ ; MS (ESI) m / z: 1299.4873 [M+H] + .

[0567] Example 23: Preparation of Compound-23

[0568] The specific synthesis operation is as follows.

[0569] Step 23-1: Preparation of M59

[0570] Following the synthesis method of M4, M54 was used to replace M3 and reacted with MMAE to produce M59, a white solid powder, in an approximately 87% yield. MS (EI) m / z: 708.95 [M+2H] 2+ ; MS (ESI) m / z: 1416.843 [M+H] + .

[0571] Step 23-2: Preparation of M60

[0572] Following the synthesis of M5, M59 was substituted for M4 and the Fmoc protecting group was removed in a piperidine-DMF solution to obtain M60, a white solid powder in approximately 68% yield. The product is hygroscopic and was used directly in the next step. MS (EI) m / z: 597.84 [M+2H] 2+ ; MS (ESI) m / z: 1194.7512 [M+H] + .

[0573] Step 23-3: Preparation of M61

[0574] Following the synthesis of compound 1, M60 was substituted for M5 and acylated with M12 under the catalysis of HATU to obtain M61 as a white solid powder in a yield of approximately 49%. MS (EI) m / z: 774.42 [M+2H] 2+ ; MS (ESI) m / z: 1547.8262 [M+H] + .

[0575] Step 23-4: Preparation of Compound-23

[0576] Following the synthesis method of compound 21, M61 was used to replace M57 and the Boc protecting group was removed in formic acid solution to obtain compound 23 as a white glassy solid in a yield of approximately 43%. MS (EI) m / z: 724.43 [M+2H] 2+ ; MS (ESI) m / z: 1447.7741 [M+H] + .

[0577] Example 24: Preparation of Compound-24

[0578] The specific synthesis operation is as follows.

[0579] Step 24-1: Preparation of Compound-24

[0580] Compound 23 (45 mg, ~30 μM) was dissolved in DMF (10 mL). After complete dissolution, a DMF solution of M36 (20 mg, 60 μM) (5 mL) was added. After stirring, a catalytic amount of DIPEA (10 μL) was added and the mixture was stirred overnight at room temperature. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting oily residue was further purified by silica gel column chromatography to afford compound 24 as a waxy white solid (31 mg, approximately 62% yield). MS (EI) m / z: 833.41 [M+2H] 2+ ; MS (ESI) m / z: 1665.8876 [M+H] + .

[0581] Example 25: Preparation of Compound-25

[0582] The specific synthesis operation is as follows.

[0583] Step 25-1: Preparation of M62

[0584] Following the synthesis of M59, MMAF was used to replace MMAE and react with M54 to produce M62, a white solid powder with a yield of approximately 68%. MS (ESI) m / z: 1430.8232 [M+H] + .

[0585] Step 25-2: Preparation of M63

[0586] Following the synthesis of M5, M62 was substituted for M4 and the Fmoc protecting group was removed in a piperidine-DMF solution to obtain M63 as a white solid powder in approximately 59% yield. It was used directly in the next step. MS (ESI) m / z: 1208.7549 [M+H] + .

[0587] Step 25-3: Preparation of M64

[0588] Following the synthesis method of M61, M63 was used to replace M60 and acylated with M12 under the catalysis of HATU to obtain M64 as a white solid powder in a yield of approximately 53%. MS (ESI) m / z: 1561.8053 [M+H] + .

[0589] Step 25-4: Preparation of Compound-25

[0590] Following the synthesis of compound 23, M64 was used to replace M61 and the Boc protecting group was removed in formic acid solution to obtain compound 25 as a white glassy solid in a yield of approximately 37%. MS (EI) m / z: 724.43 [M+2H] 2+ ; MS (ESI) m / z: 1461.7533 [M+H] + .

[0591] Example 26: Preparation of Compound-26

[0592] The specific synthesis operation is as follows.

[0593] Step 26-1: Preparation of Compound-26

[0594] According to the synthesis method of compound-24, compound-21 was used to replace compound-23 and acylated with M36 to obtain compound-26 as a white solid powder with a yield of approximately 47%. MS (ESI) m / z: 1383.5443 [M+H] + .

[0595] Example 27: Preparation of Compound-27

[0596] The specific synthesis operation is as follows.

[0597] Step 27-1: Preparation of Compound-27

[0598] Following the synthesis method of Compound-26, acetyl octapolysarcosine was substituted for M36 and acylated with Compound-21 to produce Compound-27, an off-white solid powder, in approximately 61% yield. MS (ESI) m / z: 1775.7363 [M+H] + .

[0599] Example 28: Preparation of Compound-28

[0600] The specific synthesis operation is as follows.

[0601] Step 28-1: Preparation of Compound-28

[0602] Compound 21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL) and sonicated until completely dissolved. The mixture was then cooled to 0°C in an ice bath. A catalytic amount of KCO was added, and a tetrahydrofuran solution of MeI (12 mg, 0.086 mM) (10 mL) was slowly added dropwise. The mixture was stirred at low temperature for 1 hour, and the solvent was removed by concentration under reduced pressure. The resulting residue was purified by C-18 reverse-phase chromatography to yield compound 28 as an off-white solid powder (14.5 mg, approximately 28% yield). MS (ESI) m / z: 1193.4611 [M+H] + .

[0603] Example 29: Preparation of Compound-29

[0604] The specific synthesis operation is as follows.

[0605] Step 29-1: Preparation of Compound-29

[0606] Compound-21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL), sonicated to dissolve completely, and then placed in an ice bath to cool to 0°C. P(O)(OEt)2Cl (0.045 mM) was added dropwise, and the mixture was stirred at low temperature for 1 hour, then transferred to room temperature and stirred for another 2 hours. After the reaction was completed, hydrochloric acid (10 eq) was added and the reaction was continued at room temperature for 3 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the resulting residue was purified by C-18 reverse phase chromatography to prepare compound-29, an off-white solid powder, 17 mg, with a yield of approximately 32%. MS (ESI) m / z: 1245.3962 [M+H] + .

[0607] Example 30: Preparation of Compound-30

[0608] The specific synthesis operation is as follows.

[0609] Step 30-1: Preparation of Compound-30

[0610] 4-(β-D-pyranosylamino)-4-oxobutanoic acid (CAS No.: 896730-79-3) was prepared according to the method described in the reference (Carbohydrate Research (2006), 341(8), 947-956.) with a yield of approximately 57%. It was used directly in the next reaction without long-term storage. Then, according to the synthesis method of compound-26, the prepared 4-(β-D-pyranosylamino)-4-oxobutanoic acid was used to replace M36 and acylated with compound-21 to prepare compound-30 as a white solid powder with a yield of approximately 45%. MS (ESI) m / z: 1426.5133 [M+H] + .

[0611] Example 31: Preparation of Compound-31

[0612] The specific synthesis operation is as follows.

[0613] Step 31-1: Preparation of Compound-31

[0614] Compound 21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL) and sonicated until completely dissolved. Pyridine (2 eq) was added, and the mixture was cooled to 0°C in an ice bath. ClSO₃H₃ (1 eq) was slowly added dropwise. The reaction was stirred at low temperature for 1 hour, then transferred to room temperature and stirred for an additional 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by C-18 reverse-phase chromatography to produce compound 31 as an off-white solid powder (15 mg, approximately 28% yield). MS (ESI) m / z: 1245.3857 [M+H] + .

[0615] Example 32: Preparation of Compound-35

[0616] The specific synthesis operation is as follows.

[0617] Step 35-1: Preparation of M65

[0618] 1-Amino-3,6,9,12-tetrahydropentadecan-15-oic acid (5 g, 18.85 mM) was dissolved in 50 mL of saturated NaHCO₃ and dispersed evenly. The reaction system was then placed in an ice bath to cool. 2,5-Dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester (3.22 g, 20.73 g) was added and the reaction was stirred at low temperature for half an hour before being transferred to room temperature and allowed to react for 2 hours. The pH of the reaction solution was adjusted to ~2 with HCl, and the reaction solvent was removed by concentration. The solution was redissolved in methanol and then mixed with crude silica gel. The sample was eluted with ethyl acetate to obtain the product M65 as a colorless oil, 5.31 g, with a yield of approximately 75%. MS (EI) m / z: 346.16 [M+H] + ; MS (EI) m / z: 368.15 [M+Na] + .

[0619] Step 35-2: Preparation of M66

[0620] M65 (5 g, 14.48 mM) was dissolved in tetrahydrofuran (40 mL), and NHS (2.17 g, 18.82 mM) and DCC (3.88 g, 18.82 mM) were added sequentially. The reaction mixture was stirred at low temperature for half an hour and then transferred to room temperature for another 2 hours. After the reaction was completed, the insoluble DCU was removed by filtration, and the filtrate was concentrated to obtain a light yellow oily residue. M66 was further purified by silica gel column chromatography to obtain a light yellow viscous oil (4.70 g, yield approximately 74%). MS (EI) m / z: 443.19 [M+H] + ; MS (EI) m / z: 465.20 [M+Na] + .

[0621] Step 35-3: Preparation of Compound-35

[0622] Following the synthesis method of compound-14, M66 was used instead of M16 to undergo condensation reaction with M42 to obtain compound-35 as an off-white solid powder with a yield of approximately 50%. MS (EI) m / z: 757.74 [M+2H] 2+ ; MS (ESI) m / z: 1514.7115 [M+H] + .

[0623] Example 33: Preparation of Compound-39

[0624] The specific synthesis operation is as follows.

[0625] Step 39-1: Preparation of Compound-39

[0626] M70 was prepared according to the method described in reference (WO2023125530A1; P44, compound 2i). Following the synthesis of compound 36, M12 was substituted for McOSu and reacted with M70 to produce compound 39, a light yellow solid powder, in approximately 39% yield. MS (EI) m / z: 875.38 [M+2H] 2+ ; MS (ESI) m / z: 1749.8582 [M+H] + .

[0627] Example 34: Preparation of Compound-40

[0628] The specific synthesis operation is as follows.

[0629] Step 40-1: Preparation of Compound-40

[0630] M71 was prepared according to the method described in reference (WO2023125530A1; P84, compound 27h). Following the synthesis of compound 39, M71 was substituted for M70 and reacted with M12 to yield compound 40, a light yellow solid powder, in approximately 36% yield. MS (EI) m / z: 734.77 [M+2H]. 2+ ; MS (ESI) m / z: 1468.4982 [M+H] + .

[0631] Example 35: Structure and Preparation of Compound-41

[0632] The specific synthesis operation is as follows.

[0633] Step 41-1: Preparation of M72

[0634] Benzyloxyacetic acid (46.29 mg, 0.279 mM) was dissolved in DMF (8 mL), followed by the addition of HATU (105.92 mg, 0.279 mM) and DIPEA (36 mg, 0.279 mM). After complete dissolution of the solids, the reaction system was cooled in an ice bath. MMAE (100 mg, 0.139 mM) was added, and the reaction was allowed to proceed at low temperature for 1 hour. The reaction was then slowly warmed to room temperature and continued for 4 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain an oily residue. The residue was purified by C-18 reverse-phase column chromatography and further lyophilized to obtain M72 as a white solid powder (73 mg, approximately 61% yield). MS (EI) m / z: 866.80 [M+H]. + .

[0635] Step 41-2: Preparation of Compound-41

[0636] M72 (70 mg, 0.081 mM) was dissolved in methanol (15 mL). After complete dissolution, 10% palladium on carbon (0.5 eq) was added and the reaction was stirred at room temperature overnight under a hydrogen atmosphere. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by C-18 reverse-phase column chromatography and further lyophilized to obtain compound 41 as a white solid powder (60 mg, approximately 96% yield). MS (EI) m / z: 776.75 [M+H] + ; MS (EI) m / z: 798.66 [M + Na] + .

[0637] Example 36: Preparation of Antibody-Drug Conjugates

[0638] During the preparation of ADC, the ADC antibodies selected were the humanized anti-HER-2 IgG antibody trastuzumab and the humanized anti-B7-H3 (CD276) IgG antibody CE4-Z3 to verify the coupling performance, drugability, and in vitro and in vivo efficacy characteristics of the prepared new linker-payload.

[0639] Step 36-1: General preparation method of DAR4 ADC

[0640] ① Antibody Replacement: Prior to conjugation, the selected antibody (IgG) was desalted using an AKTA system equipped with a G25 dextran gel column. The protein was then replaced with the conjugation buffer (PBS: 20mM; EDTA: 2mM; pH 6.5-8.0, or L-HIs: 20mM; EDTA: 2mM; pH 6.5-8.0). The protein absorbance was measured using a UV spectrophotometer, and the antibody concentration after the buffer exchange was calculated using the extinction coefficient method. If the antibody concentration was lower than the conjugation concentration, ultrafiltration was performed to increase the protein concentration.

[0641] ② Preparation of coupling reaction solution: According to the amount of antibody to be coupled (1 eq), use a pipette to accurately transfer the coupling system buffer corresponding to the antibody so that the antibody concentration is the initial coupling concentration, usually 1-10 mg / mL.

[0642] ③ Antibody reduction: Add 2.0-2.5 eq of TCEP·HCl solution (1.0 mg / mL-5.73 mg / mL) to the reaction vial containing the antibody solution under slow stirring. After addition, stir slowly at room temperature and react for 60-180 min.

[0643] ④ Antibody coupling: Calculate the volume of organic solvent (DMAC or DMSO) to be added so that it accounts for 5% to 15% of the total volume (usually ≤10%); at the same time, calculate the mass of the small molecule load (Linker-Payload) to be added. Usually, the small molecule load needs to be slightly excessive (usually 8eq), and then calculate the concentration of the organic solution of the load to be added. After accurately preparing the load solution, slowly add it dropwise to the reduced antibody reaction solution. Continue to stir slowly at room temperature and react for 0.25 to 1.0 hours depending on the specific coupling situation.

[0644] ⑤ Quenching of the reaction: After the reaction solution reaches the predetermined coupling time, add an excess of water-soluble small molecule N-acetylcysteine ​​(NAC) solution (1.0-3.26 mg / mL) containing a reducing thiol group, and continue the reaction with slow stirring for 15-60 minutes.

[0645] ⑥ ADC product purification: After the coupling reaction is quenched, the reaction solution is filtered and then desalted using an AKTA system equipped with a G25 dextran gel column. The front-end fraction (approximately 80%) is collected and concentrated again by ultrafiltration. After sterile filtration, the sample is aliquoted. Except for a portion of ADC samples reserved for analysis and stored at 4°C for short-term storage, the remaining ADC products are stored at -80°C until use.

[0646] Step 36-2: General preparation method of DAR8 ADC

[0647] Following the procedure in step 36-1, adjust the amount of TCEP·HCl in the antibody reduction step (③) to 4.0–8.0 eq, and adjust the amount of NAC in the linker-payload coupling step (④) and the reaction quenching step (⑤) to 8–12 eq. This yields an ADC product with a DAR of ≈ 8.0.

[0648] Step 36-3: General method for analyzing ADC DAR values

[0649] ① DAR analysis of ADCs with MMAE as the effective load

[0650] ADCs with varying drug amounts per antibody were separated at room temperature using a butyl HIC column (TSK Gel Butyl NPR 4.6×35 mm 2.5 μm, Tosoh Bioscience). HIC was also performed on an Agilent 1260 Infinity II HPLC system or a Shimadzu HPLC system with UV detection at 280 nm. Mobile phase A consisted of 1.5 mmol / L (NH₄)₂SO₄, 50 mmol / L K₂HPO₄, pH 7.0, and mobile phase B consisted of 21.3 mmol / L KH₂PO₄, 28.6 mmol / L K₂HPO₃, 25% (v / v) isopropanol, pH 7.0. The gradient program was as follows: B%: 0% to 25% (0-1 min, 0.8 mL / min), 25% (1-3 min, 0.6 mL / min). The chromatographic peak areas corresponding to DAR=0, DAR=2, DAR=4, DAR=6, and DAR=8 were integrated and weighted to calculate the DAR value of the measured ADC.

[0651] ②DAR analysis of ADCs with Dx-8951f or Dxd as payload

[0652] The absorbance of the ADC to be tested at wavelengths of 280nm and 360nm was measured using an ultraviolet spectrophotometer, and the measurement was repeated three times in parallel. The average absorbance at the two wavelengths was then calculated. Since the antibody has no ultraviolet absorption at the characteristic absorption wavelength of 360nm of the linker-payload, the standard curve method was used to calculate the relationship between the mass concentration of the linker-payload and A360 (a linear equation), as well as the multiple relationship between A360 and A280. The mass concentration of the linker-payload in the ADC and the A280 value of the linker-payload were calculated using the calculated A360. Finally, according to the Lambert-Beer law, the total absorbance at any given wavelength is equal to the sum of the absorbances of all light-absorbing chemicals present in the system at that wavelength, and the A mAb =A 平均 -A 连接子-有效荷载 Calculate the absorbance value A corresponding to the antibody at A280 mAb Finally, the DAR value of the ADC was calculated by calculating the molar ratio of the linker-payload to the antibody in the solution.

[0653] ③ ADC-DAR value analysis by LC-MS

[0654] Take 100 μg of ADC or naked antibody sample, add 1 μl of PNGaseF 500U, and digest at 37℃ overnight to obtain the sample to be tested. LC-MS parameter setting: The liquid phase system uses ACQUITY UPLC H-Class (Waters), and the chromatographic column is ACQUITY UPLC Protein, BEH SEC, The column was 1.7 μm, 2.1 mm × 100 mm, with a column temperature of 25°C and a mobile phase of 100 mM ammonium acetate in water, isocratic elution, and a flow rate of 0.1 ml / min. The mass spectrometer detection system was a Xevo G2-XS Qtof (Waters), with positive ionization and full scan detection. Data were acquired using MassLynx 4.1 (Waters) software and deconvoluted using MaxEnt I. The model parameters were set to a resolution of 2-3.5 Da. The minimum intensity ratio was set to 60%, and the output resolution was set to 1 Da. The algorithm iteration parameter was set to 20. The DAR value of the ADC was calculated by weighting the TIC peak heights corresponding to DAR = 0, DAR = 2, DAR = 4, DAR = 6, and DAR = 8 of the deconvoluted ADC.

[0655] Step 36-4: General Method for ADC Polymerization Analysis

[0656] Aggregation of the ADC products was assessed by size-exclusion chromatography, performed on an Agilent 1260 Infinity II HPLC system or a Shimadzu HPLC system with UV detection at 280 nm. The column used was a TSKgel G3000SWXL column (Tosoh Bioscience). Samples were injected at a loading of 20 to 50 μg. The mobile phase consisted of 200 mmol / L sodium phosphate and 150 mmol / L sodium chloride (pH 7.0). In addition, 10% (v / v) isopropanol was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth. The column temperature was set to room temperature. The peak areas of the corresponding monomer, fragment, and aggregate peaks in the SEC chromatograms of the tested ADCs were integrated, and the percentage of monomer content of the tested ADCs was calculated by area normalization.

[0657] The structure of the prepared ADC is shown in the following table:

[0658] in,

[0659] Denotes trastuzumab or CE4-Z3.

[0660] in,

[0661] Indicates trastuzumab or CE4-Z3.

[0662] Example 37: Preparation of Antibody-Drug Conjugates

[0663] A humanized heavy-chain camelid antibody (HCAb) against B7-H3 (CD276) was used to replace the traditional IgG antibody in Example 36 and conjugated to the novel linker-payload to prepare small antibody-HCAb-drug conjugates (HDCs) to verify the universal applicability of the linker-payload. The conjugation method and post-conjugation characterization were performed as described in Example 36.

[0664] To compare with traditional linker-payload structures, MC-VC-PABC-MMAE (Cas No.: 646502-53-6) and MC-GGFG-Dxd (Cas No.: 1599440-13-7) were purchased commercially through the Inocare reagent platform and directly conjugated to proteins. MC-VC-PABC-MMAE and MC-GGFG-Dxd are designated Compound 42 and Compound 43, respectively, in this invention. The chemical structures of MC-VC-PABC-MMAE and MC-GGFG-Dxd are as follows:

[0665] The structure of the prepared HDC is as follows:

[0666] in,

[0667] Indicates CA2-VHH25.

[0668] Example 38: Hydrophilicity / Hydrophobicity Study of Antibody-Drug Conjugates

[0669] In order to directly compare the hydrophilicity / hydrophobicity differences between the ADC based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker in the present invention and the ADC based on the traditional succinimide linker, the present invention first carried out a comparative study containing a 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and a traditional succinimide linker by using a simpler HCAb conjugate instead of an IgG conjugate.

[0670] (I) Hydrophilicity / hydrophobicity analysis of HDC based on HCAb coupling

[0671] Under the same coupling conditions, a higher hydrophobicity generally leads to higher ADC aggregation. When the DAR values ​​are roughly equivalent, the higher the hydrophilicity of the linker-payload, the higher the monomer content of the coupled product. The present invention first tests the degree of polymerization (DP) of the prepared HDC to preliminarily determine the hydrophilicity / hydrophobicity of the prepared linker-payload. Generally, a monomer content of no less than 95% is required after coupling.

[0672] Analyzing the data in the above table, we can see that:

[0673] ① By comparing the polymerization degree data of HDC-14E ​​and HDC-14F, it can be seen that reducing the DAR value of HCAb can increase the monomer content of HDC.

[0674] ② By comparing the polymerization degree data of HDC-14E ​​and HDC-35E, it can be seen that ADC based on 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has higher hydrophilicity than ADC based on traditional succinimide linker.

[0675] (II) HIC analysis based on HCAb-coupled HDC

[0676] Typically, in hydrophobic interaction chromatography (HPLC-HIC), the components of an antibody-drug conjugate (HDC) conjugated to different payloads will elute in order of hydrophilicity (naked antibody (DAR = 0) has the shortest retention time, followed by components with DAR = 2 and DAR = 4). Therefore, the hydrophobicity of the corresponding linker-payload can be determined based on the retention time of the HDC on HPLC-HIC.

[0677] Analysis of the data in the above table shows that: when the DAR values ​​are basically the same (DAR = 4), HDC-14E ​​based on the new 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has a retention time on HPLC-HIC that is significantly closer to that of the naked antibody (DAR = 0) than HDC-35E based on the traditional succinimide linker, demonstrating that the linker based on the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has higher hydrophilicity than the linker based on the traditional succinimide linker.

[0678] Example 39: Stability Study of ADC

[0679] Improved ADC stability based on new connector

[0680] To validate the stability characteristics of ADCs based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker, we compared these ADCs with traditional succinimide linkers. We selected ADC-14B and ADC-35B as model ADC molecules and conducted stability studies in human and mouse plasma. The linkers of the ADCs evaluated all contained a PEG moiety, and the stability of the ADC linker was assessed by measuring the residual antibody-conjugated drug (acDrug) content over time in plasma. The results are shown in Figures 2 and 3.

[0681] The stability test results of the above-mentioned ADC-14B and ADC-35B in human plasma and mouse plasma showed that using the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker developed by the present invention instead of the traditional succinimide linker at the antibody-terminal linker position in the linker can significantly increase the content of the antibody-conjugated drug (P values ​​are 0.0006 and 0.0124, respectively). In other words, the ADC based on the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has a lower linker-payload shedding rate in plasma.

[0682] Example 40: In vitro cytotoxicity study of ADC

[0683] The present invention is based on the screening and matching of more reasonable linker-payload combinations based on a novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker.

[0684] The present invention evaluated the in vitro activity of free MMAE, Dxd, Compound-41, and staurosporine (used as a control) on the above-mentioned tumor cell lines. Regarding the selection of cell lines, HER2-high-expressing cell lines included breast cancer cell lines BT-474, SK-BR-3, and gastric cancer cell line NCI-N87; HER2-moderate-expressing cell lines included MDA-MB-453; HER2-low-expressing cell lines included MCF-7; and HER2-null-expressing cell lines included MDA-MB-231.

[0685] As shown in Figure 4, the order of activity of the free-form payloads is: MMAE > Dxd > Compound-41. Since the EC50 value of free Compound-41 is significantly lower than that of Dxd, and the in vitro activity of the ADC (DAR4) obtained after conjugation with Compound-41 is relatively small compared to the Dxd-based ADC (DAR8), Compound-41 in this invention has the potential to be used as an ADC payload to extend the therapeutic window.

[0686] The present invention is based on a novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker to prepare compounds-1 to -31, as well as compounds-39 and -40, and couple them with various types of antibodies (taking anti-HER2 and anti-CD276 IgG antibodies, and / or HCAbs as examples) to prepare antibody-drug conjugates (ADCs or HCAbs), which are then used to carry out further drugability studies.

[0687] Example 41: In vivo efficacy study of ADC in mouse model

[0688] In order to verify the in vivo efficacy of the antibody-drug conjugates prepared by the prepared novel linker-payload, the present invention selected the antibody-drug conjugates corresponding to the humanized IgG1 antibody CE4-Z3 (abbreviated as Z3) and the humanized HCAb antibody CA2-VHH25 (abbreviated as VHH25) against B7-H3, and evaluated the in vivo efficacy characteristics of these ADCs (including HDC) in the human lung cancer Calu-6 xenograft CDX model with high B7-H3 expression.

[0689] The purpose of this in vivo efficacy study is to study the anti-tumor effect of the tested ADC in a female mouse model of subcutaneous xenografts of human lung cancer Calu-6 cell lines. The test animals used were female Balb / c-nude mice aged 6 to 8 weeks, which were raised in an environment with a temperature of 20 to 26°C and a humidity of 40 to 70%, and each test group / box was raised separately. Calu-6 cells were passaged in a culture medium of MEM (+0.01mM NEAA) + 10% FBS, and cells in the exponential growth phase were collected and resuspended to an appropriate concentration for subcutaneous tumor inoculation in mice. The experimental mice were inoculated subcutaneously with 5×10 6 Calu-6 cells were grown until the tumors grew to an average volume of approximately 100 mm 3 The mice were randomly divided into groups according to the tumor size and weight (using StudyDirector TM The mice were grouped using the software (version 3.1.399.19, supplied by Studylog System, Inc., San Francisco, CA, USA). The mice were then given the drug according to the set dose on the day of grouping. The drug volume was calculated based on V = 10 μL / g. The tumor volume was calculated as V (mm 3 )=1 / 2×(long diameter×short diameter 2) were calculated, and animal body weights were measured twice weekly. The effects of the test drug on the animals' normal behavior were observed and recorded during each measurement of tumor volume and mouse body weight. Data are presented as mean ± SD, and statistical analysis was performed using an unpaired two-tailed t-test, with a significance level set at p < 0.05. Statistical analysis was performed using GraphPad software (Version 8.0.2, La Jolla, CA, USA).

[0690] The present invention has carried out an in vivo efficacy study of ADC-14B. The results showed that compared with the vehicle group, ADC-14B had significant in vivo anti-tumor efficacy at all doses (P<0.0001). At the same time, the in vivo anti-tumor efficacy of ADC-14B at low, medium and high doses (1.5mpk, 3.0mpk and 6.0mpk) showed a significant dose-effect dependence. Among them, compared with the 3.0mpk dose group, the 1.5mpk dose group had a significant advantage in in vivo efficacy (P=0.0092); compared with the 6.0mpk dose group, the 6.0mpk dose group had a significant advantage in in vivo efficacy (P=0.0028); compared with the 6.0mpk dose group, the 6.0mpk dose group had a significant advantage in in vivo efficacy (P=0.0428). The results are shown in Figure 5.

[0691] Commercially available MC-GGFG-Dxd (Compound No. 43) was conjugated with DS7300 from Daiichi Sankyo Co., Ltd. to produce an ADC with a DAR of 4 (Code: DS7300-43). Conversely, a nonspecific isotype IgG1 was conjugated with Compound 14 to produce an ADC with a DAR of 4 (Code: IgG-14B).

[0692] Furthermore, the present invention compared the in vivo efficacy characteristics of ADC-14B, DS7300-43, and IgG-14B in a xenograft mouse model of PC3 prostate cancer cells with moderate expression of the antigen B7-H3. As shown in FIG6A , ADC-14B exhibited significant in vivo anti-tumor efficacy at all doses compared to the vehicle group (P < 0.0001). Furthermore, the in vivo anti-tumor efficacy of ADC-14B at low, medium, and high doses (1.5 mpk, 3.0 mpk, and 6.0 mpk) exhibited a clear dose-response relationship. Specifically, the in vivo efficacy of the 1.5 mpk dose group was significantly superior to that of the 3.0 mpk dose group (P = 0.0038); and the in vivo efficacy of the 6.0 mpk dose group was significantly superior to that of the 3.0 mpk dose group (P = 0.0356). On the other hand, as shown in FIG6B , at the same dose (3.0 mpk), ADC-14B had a significant in vivo efficacy advantage over DS7300-43 and IgG-14B (P values ​​were 0.0076 and <0.0001, respectively).

[0693] Compound 14 of the present invention was conjugated with the anti-B7-H3 humanized HCAb CA2-VHH25 screened by our team to produce HDC-14E ​​with a DAR of ≈ 4. In vivo efficacy results showed that compared with the vehicle group, tumor growth was significantly inhibited in all HDC-14E ​​(DAR4) test groups (P < 0.0001). Furthermore, the anti-tumor efficacy of HDC-14E ​​(DAR4) at low, medium, and high doses (1.5 mpk, 3.0 mpk, and 6.0 mpk) in vivo showed a clear dose-dependent relationship. Among them, the 1.5 mpk dose group showed a significant advantage over the 3.0 mpk dose group (P=0.0001); the 1.5 mpk dose group showed a significant advantage over the 6.0 mpk dose group (P<0.0001); and the 3.0 mpk dose group showed a significant advantage over the 6.0 mpk dose group (P<0.0001). The results are shown in Figure 7.

[0694] Example 42: Safety Study of Anti-B7-H3 ADC in Mouse Model

[0695] To directly compare the safety of the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker-based ADCs in the present invention with those based on traditional succinimide linkers, the present invention used ADC-14B (DAR4) based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and ADC-35B (DAR4) based on the traditional succinimide linker as model ADC molecules. A preliminary comparison of their tolerability at a high dose (200 mg / kg) was conducted in B-hB7-H3 (C57BL6-Cd276tm1 (CD276) / Bcgen, BIOCYTOGEN, Beijing, CN) transgenic mice. Each test group contained three animals.

[0696] The test results showed that none of the test animals died 4 days after administration, and all the test animals did not show serious physical signs or behavioral abnormalities.

[0697] Four days after administration, the average body weight of the animals in the ADC-14B group decreased by 11.0% compared with before administration, while the average body weight of the animals in the ADC-35B group decreased by 18.4% compared with before administration. The percentage of body weight loss in the ADC-14B group (9.4%, 9.4%, 14.4%) was significantly different from the percentage of body weight loss in the ADC-35B group (18.6%, 19.5%, 17.1%) (P=0.0154).

[0698] After 7 days of administration, the average body weights of the ADC-14B and ADC-35B test animals recovered to 98.53% and 68.73% of their pre-dose levels, respectively. The percentage of body weight loss in the ADC-14B group (-3.53%, 1.56%, 6.38%) was significantly different from that in the ADC-35B group (32.79%, 31.61%, 29.41%) (P = 0.0006).

[0699] In addition, serious adverse reactions were observed in the animals in the ADC-35B group, such as unstable gait, dull fur, perianal dirtiness, and slow movements, while no obvious abnormalities were observed in the animals in the ADC-14B group.

[0700] The above data show that the dose of 200 mg / kg has not yet reached the maximum tolerated dose of ADC-14B, while the maximum tolerated dose of ADC-35B is less than 200 mg / kg, proving that ADC-14B based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has potential safety advantages compared with ADC-35B based on the traditional succinimide linker.

[0701] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or isotopic variant thereof: Wherein, R1 is selected from C 1-6 alkyl or C 1-6 haloalkyl; W1 is selected from -O-, -S-, -NR b -, -C(O)O-, -C(O)NR b -, -O-C(O)-, -NR b -C(O)-, -S(O) p O- or -O-S(O) p -; wherein p = 1 or 2; L1 is a chemical bond or -(CH2) m1 -(OCH2CH2) n1 -(CH2CH2O) n2 -(CH2) r1 -(L) q -(CH2) r2 -(OCH2CH2) n3 -(CH2CH2O) n4 -(CH2) m2 -; wherein -L- is selected from -O-, -NR b -, -C(O)NR b -, -C(O)O-, -NR b -C(O)-, -O-C(O)-, -C 3-8 subcycloalkyl-, -3- to 8-membered heterocycloalkyl-, -C 6-10 arylene- or 5- to 10-membered heteroarylene; each of m1, m2, n1, n2, n3, n4, r1 and r2 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; q = 0, 1 or 2; W2 is selected from a chemical bond, -O-, -S-, -NR b -, or -C(O)-; R2 is selected from H, D, halogen, -OR a , -NR b R c or the following groups: R3 is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl; s = 0, 1 or 2; wherein R a , R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclic group, C 6-10 aryl or 5- to 10-membered heteroaryl; or R b and R c together with the N atom to which they are attached form a 3- to 10-membered heterocyclic group; wherein the above groups are optionally substituted with one or more deuteriums until fully deuterated.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, R1 is selected from C 1-4 alkyl or C 1-4 haloalkyl, preferably methyl.

3. A compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, W1 is selected from -C(O)O-, -C(O)NR b -, -O-C(O)- or -NR b -C(O)-, preferably -C(O)O- or -C(O)NR b -.

4. The compound of formula (I) according to any one of claims 1-3, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, L1 is selected from a chemical bond, -(CH2) m1 -(CH2CH2O) n1 -(CH2) r1 -L-(CH2) r2 -(CH2CH2O) n2 -(CH2) m2 - or -(CH2) m3 -(OCH2CH2) n3 -(CH2) r3 -L-(CH2) r4 -(OCH2CH2) n4 -(CH2) m4 -; Preferably, L1 is selected from a chemical bond, -(CH2CH2O) n5 -(CH2) r5 -L-(CH2) m5 -、-(OCH2CH2) n6 -(CH2) r6 -L-(CH2) m6 -、-(CH2) m7 -L-(CH2CH2O) n7 -(CH2) r7 - or -(CH2) m8 -L-(OCH2CH2) n8 -(CH2) r8 -; Preferably, L1 is selected from a chemical bond, -(CH2) m9 -(CH2CH2O) n9 -(CH2) r9 - or -(CH2) m10 -(OCH2CH2) n10 -(CH2) r10 -; Preferably, L1 is selected from a chemical bond, -(CH2CH2O) n11 -(CH2) m11 -、-(CH2) m12 -(CH2CH2O) n12 -、-(OCH2CH2) n13 -(CH2) m13 - or -(CH2) m14 -(OCH2CH2) n14 -; Preferably, L1 is selected from a chemical bond, -(CH2) m15 -O-(CH2) m16 -, -(CH2) m17 -O-, -(CH2) m18 -NH-, -O-(CH2) m19 - or -NR b -(CH2) m20 -; preferably, L1 is selected from a chemical bond, -(CH2) m21 -, -L-, -(CH2) m22 -L- or -L-(CH2) m23 -; Preferably, L1 is selected from a chemical bond, -(CH2CH2O) n11 -(CH2) m11 - or -(CH2) m21 -; wherein m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, m12, m13, m14, m15, m16, m17, m18, m19, m20, m21, m22, m23 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n1, n2, n3, n4, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r1, r2, r3, r4, r5, r6, r7, r8, r9, r10 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

5. A compound of formula (I) according to any one of claims 1-4, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, -L- is selected from -C(O)NR b -, -C(O)O-, -NR b -C(O)- or -O-C(O)-.

6. A compound of formula (I) according to any one of claims 1-5, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, W2 is selected from a chemical bond or -C(O)-.

7. A compound of formula (I) according to any one of claims 1-6, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, R2 is selected from H, -OH or The compound of formula (I) according to any one of claims 1-7, or a pharmaceutically acceptable salt or isotopic variant thereof, having the following general formula: wherein each group is as defined in any one of claims 1-7.

9. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein, R1 is Me; W1 is -C(O)O- or -C(O)NH-; L1 is selected from a chemical bond, -(CH2CH2O) n11 -(CH2) m11 - or -(CH2) m21 -; wherein n11 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m11 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m21 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; W2 is selected from a chemical bond or -C(O)-; R2 is selected from H, -OH or 10. A compound according to any one of claims 1-9, or a pharmaceutically acceptable salt or isotopic variant thereof, wherein the compound is selected from the following:

11. Use of the compound according to any one of claims 1-10, or a pharmaceutically acceptable salt or isotopic variant thereof, in the preparation of an antibody-drug conjugate.

12. A compound of formula (II), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof: Wherein, L2 is selected from the following amino acid residues or oligopeptide residues composed of 2-10 or fewer amino acids, wherein the amino acids are selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acids are optionally substituted with 1, 2, 3, 4, 5 or 6 R4; wherein the N-terminus of the amino acid residue or oligopeptide residue is connected to W2 and the C-terminus is connected to L3; R4 is selected from D, halogen, NO2, -OR a , -NR b R c , C 1-6 alkyl, C 1-6 haloalkyl, polyethylene glycol, polyglycine, pentose, hexose, sulfonic acid group, mesyl group, phosphoric acid group, phosphorous acid group, quaternary ammonium salt, or is selected from the following groups: L3 is selected from the following structures: wherein NH is connected to L2 and C(O) is connected to D; R5 is selected from H, D, halogen, NO2, -OR a , -NR b R c , C 1-6 alkyl or C 1-6 haloalkyl; t = 0, 1, 2, 3 or 4; R6 is selected from H, D is an active compound selected from a drug, a cytotoxin, a detection reagent, a diagnostic reagent or a targeting vector; wherein the above groups are optionally substituted with one or more deuteriums until fully deuterated; other groups are as defined in any one of claims 1-10.

13. The compound of formula (II) according to claim 12, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, L2 is selected from the following amino acid residues or oligopeptide residues composed of 2 to 5 of the following amino acids, wherein the amino acids are selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine; Preferably, L2 is selected from the following amino acid residues or oligopeptide residues composed of 2 to 5 of the following amino acids, wherein the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine; Preferably, L2 is selected from dipeptide, tripeptide or tetrapeptide residues composed of the following amino acids, wherein the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine; Preferably, L2 is selected from valine-citrulline, valine-alanine, valine-lysine, phenylalanine-lysine, lysine-lysine, alanine-lysine, phenylalanine-citrulline, leucine-citrulline, isoleucine-citrulline, phenylalanine-alanine, lysine-valine-citrulline, lysine-valine-alanine, valine-lysine-glycine, glycine-valine-lysine, glycine-valine-alanine, glutamine-valine-alanine, glutamine-valine-citrulline, glutamic acid-valine-alanine, glutamic acid-valine-citrulline, alanine-alanine-alanine, alanine-alanine-asparagine, phenylalanine-phenylalanine-lysine, glycine-phenylalanine-lysine, leucine-alanine-leucine, isoleucine-alanine-leucine, valine-alanine-valine, glycine-glycine-phenylalanine-glycine, alanine-leucine-alanine-leucine and glycine-phenylalanine-leucine-glycine; Preferably, L2 is selected from glutamine-valine-alanine, glutamine-valine-citrulline, valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine; Preferably, L2 is selected from valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine.

14. The compound of formula (II) according to claim 12 or 13, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, R4 is selected from H, D, halogen, NO2, -OR a , -NR b R c , C 1-6 alkyl, C 1-6 haloalkyl, polyethylene glycol, polyglycine, pentose, hexose, sulfonic acid group, mesyl group, phosphoric acid group, phosphorous acid group, quaternary ammonium salt, or selected from the following groups:

15. The compound of formula (II) according to any one of claims 12 - 14, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, L3 is selected from the following structures: Preferably, R6 is selected from H, 16. The compound of formula (II) according to any one of claims 12 - 15, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, D is selected from cytotoxins, anti - tumor drugs, anti - infective drugs or immunomodulatory drugs; Preferably, D is a cytotoxin, such as a tubulin inhibitor, a topoisomerase inhibitor, a DNA alkylating agent, a DNA intercalator, an enzyme inhibitor, an immunomodulator, a PROTAC, an antimetabolite, an active peptide or an active nucleotide sequence; Preferably, D is selected from tubulin inhibitors, DNA topoisomerase inhibitors, drugs that interfere with DNA synthesis, DNA alkylating agents, DNA intercalators, enzyme inhibitors, RNA inhibitors, Bcl - xL inhibitors, NAMPT inhibitors, proteasome inhibitors, calicheamicin, immunomodulators, PROTACs, antimetabolites, active peptides, nucleotides, tumor signaling pathway inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, drugs acting on structural proteins, metal complexes or glycopeptide antibiotics; Preferably, D is a cytotoxin, preferably selected from maytansine and its derivatives, auristatin and its derivatives, Tubulysins and its derivatives, tubulin and its derivatives, cryptophycin and its derivatives, eribulin and its derivatives, pyrrolobenzodiazepines (PBD) and its derivatives, enediyne molecules and its derivatives, duocarmycin and its derivatives, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis; Preferably, D is selected from auristatin and its derivatives, camptothecin and its derivatives, maytansine and its derivatives, calicheamicin and its derivatives, paclitaxel and its derivatives, pyrrolobenzodiazepines and its derivatives, duocarmycin, doxorubicin, melphalan, mitomycin C, chlorambucil, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis; Preferably, D is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1, DM4, Dxd (CAS: 1599440-33-1), exatecan (Dx-8951f), oxaliplatin, bleomycin, pingyangmycin, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, actinomycin D, doxorubicin, docamicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, serine / threonine kinase inhibitor, tyrosine kinase inhibitor, aspartate kinase inhibitor or histidine kinase inhibitor; Preferably, D is selected from MMAE, MMAF, Dxd or Dx-8951f.

17. A compound of formula (II) according to any one of claims 12-16, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, having the following general formula: Wherein each group is defined as in any one of claims 1-16.

18. A compound according to any one of claims 12-17, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein R1 is Me; W1 is -C(O)O- or -C(O)NH-; L1 is selected from -(CH2CH2O) n11 -(CH2) m11 - or -(CH2) m21 -; where n11 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m11 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m21 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; W2 is -C(O)-; L2 is selected from dipeptide, tripeptide or tetrapeptide residues composed of the following amino acids, wherein the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6 R4; Preferably, L2 is selected from valine-citrulline, valine-alanine, valine-lysine, phenylalanine-lysine, lysine-lysine, alanine-lysine, phenylalanine-citrulline, leucine-citrulline, isoleucine-citrulline, phenylalanine-alanine, lysine-valine-citrulline, lysine-valine-alanine, valine-lysine-glycine, glycine-valine-lysine, glycine-valine-alanine, glutamine-valine-alanine, glutamine-valine-citrulline, glutamate-valine-alanine, glutamate-valine-citrulline, alanine-alanine-alanine, alanine-alanine-asparagine, phenylalanine-phenylalanine-lysine, glycine-phenylalanine-lysine, leucine-alanine-leucine, isoleucine-alanine-leucine, valine-alanine-valine, glycine-glycine-phenylalanine-glycine, alanine-leucine-alanine-leucine, and glycine-phenylalanine-leucine-glycine, and the amino acids are optionally substituted with 1, 2, 3, 4, 5, or 6 R4; R4 is selected from H, D, halogen, NO2, -OR a , -NR b R c , C 1-6 alkyl, C 1-6 haloalkyl, polyethylene glycol, polyglycine, pentose, hexose, sulfonic acid group, mesyl group, phosphoric acid group, phosphorous acid group, quaternary ammonium salt, or is selected from the following groups: L3 is selected from the following structures: wherein R5 is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl; t = 0, 1, 2, 3, or 4; R6 is selected from H, D is selected from tubulin inhibitors, topoisomerase I inhibitors, and DNA alkylating agents, such as MMAE, MMAF, Dxd, or Dx-8951f.

19. A compound according to any one of claims 12 - 18, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereoisomer, meso - form, racemate or tautomer thereof, wherein the compound is selected from the following:

20. A compound of formula (III), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, having the following general formula: Wherein, A is a targeting molecule; x = 1, 2, 3, 4, 5, 6, 7, or 8; The other groups are as defined in any one of claims 1-19.

21. The compound of formula (III) according to claim 20, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, A is selected from proteins, antibodies, antibody fragments, fusion proteins, polypeptides, enzymes, and small molecules; A is preferably an antibody, preferably a monoclonal antibody, such as a monospecific monoclonal antibody and a bispecific monoclonal antibody; A is preferably an IgG-type antibody and an HCAb-type antibody targeting tumor-associated antigens.

22. The compound of formula (III) according to claim 21, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein, The tumor-associated antigens targeted by A include, but are not limited to: HER2, Trop-2, Claudin-6, Claudin-9, Claudin-18.2, EGFR, c-Met, CD19, PSMA, Muc1, BCMA, PD-L1, CD33, CD30, CD22, CD79b, Nectin-4, CD19, tissue factor, FRα, B7-H3, B7-H4, CDH3, CDH6, CDH17, ALPP, CD56, CD37, HER3, ROR1, MSLN, TNF-α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM5, CEACAM6, CD138, GC-C, LIV-1, CA6, FUT3, IGF-1R, CTLA4, RNF43, DPEP3, 5T4, ITGB6, EFNA4, CD228, Notch3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, TIM1, sTn, ETB, Globo H, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, LYFD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, Muc1, LRRC15, CD44, CD70, EphA2, CXCR4, DDR1, DKL1, FOLR, CD45, DSG2, ALK, TRAIL, EpCAM, VEGFR2, CD47, CD49, SSEA-4, DCLK1, OacGD2, CD73, EN01, BSG, CD24, GLUT1 and GPRC5D, preferably HER2, HER3, EGFR, Trop-2, Claudin-6, Claudin-18.2, B7-H3, B7-H4, CDH3, CDH6, CDH17, FRα, ROR1, ALPP, CEACAM5, CEACAM6 or FOLR.

23. A compound of formula (III) according to any one of claims 20-22, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereoisomer, meso form, racemate or tautomer thereof, wherein, x = 2, 3, 4, 5, 6, 7 or 8; preferably x = 2, 4, 6 or 8; x = 4, 6 or 8.

24. A compound according to any one of claims 20 - 23, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, having the following general formula: Wherein each group is defined as in any one of claims 1-23.

25. A compound according to any one of claims 20-24, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereoisomer, meso form, racemate or tautomer thereof, wherein A is an antibody, preferably a monoclonal antibody, such as a monospecific monoclonal antibody and a bispecific monoclonal antibody; x = 2, 3, 4, 5, 6, 7 or 8; preferably x = 2, 4, 6 or 8; x = 4, 6 or 8; W1 is -C(O)O- or -C(O)NH-; L1 is selected from -(CH2CH2O) n11 -(CH2) m11 - or -(CH2) m21 -; where n11 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m11 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m21 = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; W2 is -C(O)-; L2 is selected from dipeptide, tripeptide or tetrapeptide residues composed of the following amino acids, where the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6 R4; Preferably, L2 is selected from valine-citrulline, valine-alanine, valine-lysine, phenylalanine-lysine, lysine-lysine, alanine-lysine, phenylalanine-citrulline, leucine-citrulline, isoleucine-citrulline, phenylalanine-alanine, lysine-valine-citrulline, lysine-valine-alanine, valine-lysine-glycine, glycine-valine-lysine, glycine-valine-alanine, glutamine-valine-alanine, glutamine-valine-citrulline, glutamate-valine-alanine, glutamate-valine-citrulline, alanine-alanine-alanine, alanine-alanine-asparagine, phenylalanine-phenylalanine-lysine, glycine-phenylalanine-lysine, leucine-alanine-leucine, isoleucine-alanine-leucine, valine-alanine-valine, glycine-glycine-phenylalanine-glycine, alanine-leucine-alanine-leucine and glycine-phenylalanine-leucine-glycine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6 R4; where the N-terminus of the amino acid residue or oligopeptide residue is connected to W2, and the C-terminus is connected to L3; R4 is selected from H, D, halogen, NO2, -OR a , -NR b R c , C 1-6 alkyl, C 1-6 haloalkyl, polyethylene glycol, polyglycine, pentose, hexose, sulfonic acid group, mesyl group, phosphoric acid group, phosphorous acid group, quaternary ammonium salt, or selected from the following groups: L3 is selected from the following structures: where NH is connected to L2 and C(O) is connected to D; R5 is selected from H, D, halogen, C 1-6 alkyl or C 1-6 haloalkyl; t = 0, 1, 2, 3 or 4; R6 is selected from H, D is selected from tubulin inhibitors, topoisomerase I inhibitors and DNA alkylating agents, such as MMAE, MMAF, Dxd or Dx-8951f.

26. The compound of any one of claims 20-25, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, wherein the compound is selected from: wherein, represents a monoclonal antibody, preferably an IgG1 and HCAb type monoclonal antibody against HER2 and B7-H3.

27. A pharmaceutical composition comprising the compound of any one of claims 20-26 or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof, and a pharmaceutically acceptable excipient or adjuvant.

28. Use of the compound of any one of claims 20-26 or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, meso form, racemate or tautomer thereof in the preparation of a drug for treating a disease or disorder and alleviating the severity of the disease or disorder.

29. A compound according to any one of claims 20-26, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereoisomer, meso form, racemate or tautomer thereof, or a pharmaceutical composition according to claim 27, for use in the treatment of a disease or disorder and for reducing the severity of said disease or disorder.

30. A method of treating a disease or disorder in a subject and for reducing the severity of said disease or disorder, comprising administering to the subject a compound according to any one of claims 20-26, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereoisomer, meso form, racemate or tautomer thereof, or a pharmaceutical composition according to claim 27.

31. Use according to claim 28, or use of a compound according to claim 29, or a method according to claim 30, wherein the disease or disorder is selected from tumours, cancers, autoimmune diseases, infectious diseases, haematological diseases, metabolic diseases, inflammation; Preferably, the cancer is selected from breast cancer, lung cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, gastric cancer, oesophageal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, urethral cancer, bowel cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, penile cancer, thyroid cancer, anal cancer, melanoma, brain cancer, gallbladder cancer, oesophageal cancer, bile duct cancer, head and neck cancer, lymphoma, multiple myeloma, blastoma, leukaemia, myeloma, plasmacytoma, sarcoma, and pancreatic cancer, mesothelioma, nasopharyngeal cancer.