Bifunctional linker compound, antibody drug conjugate, preparation method for antibody drug conjugate, and use of antibody drug conjugate

Abstract

The present application relates to a bifunctional linker compound, an antibody drug conjugate, a preparation method for an antibody drug conjugate, and a use of an antibody drug conjugate. The bifunctional linker compound is a compound represented by formula (I), or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof. All groups in the formula are defined in the description. The compound of the present application not only can be used for preparing an antibody drug conjugate having improved homogeneity, but also can improve the structural stability of an antibody. M-L-D (I)

Description

Bifunctional linker compound, antibody-drug conjugate, and preparation method and application thereof

[0001] This application claims priority to Chinese patent application No. 2023109887077, filed on August 8, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of antibody-drug conjugates. Specifically, the present invention provides a series of drug-bifunctional linker compounds, drug-antibody conjugates, and preparation methods and applications thereof. Background Art

[0003] Antibody drug conjugates (ADCs) are a new and effective targeted drug consisting of three components: an antibody, a linker, and a toxin. The toxin (payload) is the key component for drug efficacy, while the antibody plays a crucial role in targeting. The linker not only connects the two but also influences the ADC's stability, drug release mechanism, and antibody conjugation method.

[0004] Traditional ADC products utilize two conjugation methods: amide bond formation between a lysine (Lys) on the antibody and a carboxylic acid group on the payload-linker, or Michael addition of a maleimide linker to eight cysteines in four disulfide bonds within the reduced antibody. Consequently, most marketed ADCs with a drug-antibody ratio (DAR) of ~4 are mixtures of average DAR values ​​derived from random conjugation, rather than homogeneous conjugates.

[0005] Summary of the Invention

[0006] Through extensive research, the inventors have surprisingly discovered a series of bifunctional linker compounds that can crosslink with the sulfhydryl groups adjacent to cysteine ​​residues in reduced antibodies to form uniform antibody-drug conjugates. These bifunctional linker-antibody conjugates (ADCs) not only improve drug stability but also enhance efficacy, possessing significant application value in tumor treatment.

[0007] Bifunctional linkers can simultaneously connect to two chemical functional groups, such as the sulfhydryl groups of two cysteines formed by reducing a pair of disulfide bonds. Therefore, the sulfhydryl groups of eight cysteines formed by reducing four pairs of disulfide bonds can be saturated with four bifunctional linkers to form a site-specific, quantitative antibody-drug conjugate.

[0008] One aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

[0009] Another aspect of the present invention provides a drug-antibody conjugate or a pharmaceutically acceptable salt thereof.

[0010] Another aspect of the present invention provides a method for preparing a drug-antibody conjugate or a pharmaceutically acceptable salt thereof.

[0011] Another aspect of the present invention provides a bifunctional linker compound.

[0012] Another aspect of the present invention provides pharmaceutical compositions.

[0013] Another aspect of the present invention provides the use of the above-mentioned drug-antibody conjugate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer.

[0014] Another aspect of the present invention provides a method for treating cancer, comprising the step of administering to a patient in need thereof a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, a drug-antibody conjugate of the present invention, a bifunctional linker compound of the present invention, or a pharmaceutical composition of the present invention.

[0015] The present invention can not only prepare antibody-drug conjugates with improved homogeneity but also enhance the structural stability of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1 is a graph showing the tumor (pancreatic cancer) inhibitory effect of the drug-antibody conjugate in Effect Example 2.

[0017] FIG2 is a photograph showing the tumor-suppressing effect of a drug-antibody conjugate in the CFPAC-1 model.

[0018] FIG3 is a graph showing the tumor (breast cancer) inhibitory effect of the drug-antibody conjugate in Effect Example 2. FIG.

[0019] FIG4 is a photograph showing the tumor-suppressing effect of a drug-antibody conjugate in an MCF-7 model. DETAILED DESCRIPTION

[0020] I. Definition

[0021] The term "antibody" as used herein is used in the broadest sense and specifically encompasses 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 or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens typically have multiple binding sites, also known as epitopes, which are recognized by CDRs (complementary determining regions) on a variety of 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 antigen binding sites that immunospecifically bind to a target antigen or a portion thereof, including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases.

[0022] As used herein, the term "antibody fragment" includes a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the antibodies described herein that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0023] The term "monoclonal antibody" used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for the possible naturally occurring mutations that may exist in a small amount, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, unlike polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that their synthesis is not contaminated by other antibodies. The modifier "monoclonal" indicates that the antibody is a characteristic obtained from a substantially homogeneous antibody population and should not be construed as needing to produce the antibody by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by first using a hybridoma method, or can be prepared by a recombinant DNA method. Monoclonal antibodies can also be separated from phage antibody libraries.

[0024] The monoclonal antibodies herein include, in particular, "chimeric" antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. The chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0025] The term "chimeric" antibody as used herein refers to an antibody in which a portion of the heavy and / or light chains is derived from a specific source or species, while the remainder of the heavy and / or light chains is derived from a different source or species. The term "intact antibody" as used herein refers to an antibody comprising a VL domain and a VH domain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, CH2, and CH3. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc constant region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (such as B cell receptors and BCRs).

[0026] The term "antibody-drug conjugate (ADC)" used in this application refers to a small molecule drug with biological activity connected to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into target cells.

[0027] As used herein, the term "bifunctional linker" refers to a structure that can simultaneously connect two chemical functional groups (specifically, an antibody and a cytotoxic drug moiety). This structure comprises a group that connects to the antibody and a group that connects to the cytotoxic drug moiety, and its specific structure is shown as M described below.

[0028] The term "spacer unit E" used in this application is also called a self-immolatIIIe structure, which is a linking group located between L and D. The spacer unit (E) is selected from one of p-aminobenzyl carbonate amide (PAB carbamate), p-aminobenzyl carbonate diester (PAB carbonate), hemiaminal and methylenediamine.

[0029] The term "spacer unit F" used in this application refers to a connecting group located between the M and the L, and the spacer unit F is -NH-(CH2CH2-O) q -CH2CH2-C(=O)- or a single bond, wherein q represents an integer of 1 to 6.

[0030] As used herein, the term "cytotoxic drug moiety," also referred to as a "small molecule drug," refers to a compound that has a tumor cell-killing effect. Examples of the cytotoxic drug moiety include at least one of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, a DNA synthesis inhibitor, an RNA polymerase inhibitor, a splicesome inhibitor, a proteolysis-targeting chimera (PROTAC), and an immunomodulator.

[0031] Immunomodulators include but are not limited to immunosuppressants and agonists, and their targets include PD-1 / PD-L1, PD-L2, CTLA-4, LAG-3, IDO, TIM3, TIGIT, CD47, SIRPα, 4-1BB, CSF-1 / CSF1R, GITR, OX40, CD40, CD27, CD28, B7H4, B7H3, TGFβ, BTLA, VISTA, ICOS, CD39, CD73, A2AR, KIR and NKG2A, etc.; as well as cell therapies related to immunotherapy.

[0032] The small molecule drugs used in this application can be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis, trans structures) and optical isomers (such as enantiomers), and are therapeutic substances composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds containing asymmetric carbon atoms in this application can be separated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this application.

[0033] The small molecule drugs used in this application also include tautomers, which are formed when a single bond is exchanged with an adjacent double bond accompanied by the migration of a proton.

[0034] The term "precursor" as used herein refers to a compound that, after being administered into the human body via an appropriate route, undergoes metabolism or simple chemical changes in the patient's body to transform into the compound of the present invention, as contained in the general formula (I), and the corresponding salt. Precursors of compounds include, but are not limited to, various carboxylates, carbonates, phosphates, sulfates, sulfonates, amino acid esters, gluconates, and various amides, acetals, hemiacetals, and carbonic acid amides.

[0035] As used herein, numerical ranges refer to the individual integers within the given range. For example, "C1-C6" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms; "C3-C6" means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0036] When any variable (such as R n ) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 R, the group may be optionally substituted with up to 5 R, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0037] The term "alkyl" as used herein refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 2,2-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate groups, preferably methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl.

[0038] As used herein, the term "heteroalkyl" by itself or in combination with another term refers to a stable straight-chain, branched hydrocarbon radical or combination thereof consisting of a certain number of carbon atoms and at least one heteroatom. In a typical embodiment, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatom or heteroatom group can be located at any interior position of the heteroalkyl group, including the position at which the hydrocarbon group is attached to the rest of the molecule, and examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3. However, the terms "alkoxy", "alkylamino" and "alkylthio" are conventional expressions and refer to those alkyl groups that are attached to the remainder of the molecule through an oxygen atom, amino group or sulfur atom, respectively, "alkoxy" refers to -O-alkyl, "alkylamino" refers to -NH-alkyl, and "alkylthio" refers to -S-alkyl, wherein alkyl is as defined above.

[0039] The term "alkenyl" as used herein is an unsaturated straight-chain or branched hydrocarbon radical, in particular an alkenyl radical having 2 to 10 carbon atoms, with double bonds in any desired positions, for example C2-C6 alkenyl, such as ethenyl, 1-propenyl, 2-propenyl, 1-methyl-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, alkenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-vinyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-Methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3- butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl, and 1-ethyl-2-methyl-2-propenyl;

[0040] The term "heterocyclyl" or "heterocyclyl ring" as used herein refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen or S(O)m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1-4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms; most preferably, it contains 3 to 8 ring atoms; further preferably, it contains 3-8 membered heterocyclyls containing 1-3 nitrogen atoms, optionally substituted by 1-2 oxygen atoms, sulfur atoms, oxo groups, including nitrogen-containing monocyclic heterocyclyls, nitrogen-containing spiroheterocyclyls or nitrogen-containing fused heterocyclyls.

[0041] The term "aryl" as used herein refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 12-membered, such as phenyl and naphthyl.

[0042] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from (halo)alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydrogen, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0043] The term " alkoxy " used in this application refers to-O-(alkyl) and-O-(non-substituted cycloalkyl), wherein the definition of alkyl is as described above. The limiting examples of alkoxy comprises: methoxyl group, ethyoxyl, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or non-substituted, and when substituted, substituent group is preferably one or more following groups, and it is independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydrogen, nitro, chloro, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0044] The term "heteroaryl" as used herein is used interchangeably herein and refers to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic group having 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, wherein one or more ring atoms are optionally independently substituted with one or more substituents described below. The heterocycle may be a monocycle having 3-7 ring members (2-6 carbon atoms and 1-4 heteroatoms selected from N, O, P, and S) or a bicyclic, such as a bicyclic [4,5], [5,5], [5,6], or [6,6] system having 7-10 ring members (4-9 carbon atoms and 1-6 heteroatoms selected from N, O, P, and S). "Heterocyclyl" also includes groups wherein the heterocyclic group is fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring. Examples of heterocycles include, but are not limited to, morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S,S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholinyl, thiomorpholinyl, thiooxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxacyclohexanyl, Cyclobutane, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolane, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiolanyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolyl, quinolizinyl, and N-pyridylurea. Spirocyclic moieties are also included within the scope of this definition. Examples of heterocyclic groups wherein 2 ring atoms are substituted with oxo (=0) moieties are pyrimidinone and 1,1-dioxo-thiomorpholinyl.Heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0045] The term "substituted" used herein refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1-3 hydrogen atoms that are independently replaced by the substituents of corresponding number. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible replacement without paying too much effort. For example, amino or hydroxyl groups with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (such as olefinic) bond.

[0046] Refers to the chemical bond connection.

[0047] Although the term "optionally substituted" does not appear, if substitution occurs at any position on a ring (eg, an aliphatic ring or an aromatic ring), it should be understood that the position and number of substitution are arbitrary as long as it is chemically feasible.

[0048] The term "pharmaceutically acceptable salt" as used herein refers to a salt formed between a corresponding amine compound and an inorganic acid or organic acid, or a salt formed between a corresponding carboxylic acid compound and an alkali metal or alkaline earth metal, or a salt formed with an organic amine. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like; organic acids include, but are not limited to, acetic acid, propionic acid, butyric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, tartaric acid, and the like; alkali metal or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0049] The medicament or pharmaceutical composition of the present application can be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in a dosage unit formulation containing a conventional non-toxic pharmaceutically acceptable carrier. It is generally desirable to use the oral route. The active agent can be administered orally in the form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0050] For oral administration in the form of tablets or capsules, the active drug component can be mixed with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, and the like. For oral administration in liquid form, the drug component can be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils), a preservative (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), etc. Stabilizers such as antioxidants (BHA, BHT, propyl citric acid, sodium ascorbate, citric acid) can also be added to stabilize the dosage form.

[0051] The tablet comprising the active compound can be coated by methods well known in the art. The composition of the present application comprising the compound of formula I as the active compound can also be introduced into beads, microspheres or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). The preparation of liquid for oral administration can take the form of, for example, solution, syrup, emulsion or suspension or they can be presented as a dry product reconstituted with water or other suitable adjuvants before use. The preparation for oral administration can be suitably formulated so that the active compound is controlled or released in a delayed manner.

[0052] As used herein, the term "treating," ...

[0053] As used herein, the term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0054] The term "pharmaceutical composition" as used herein refers to a composition comprising the compound described herein or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0055] The term "tumor" or "cancer" as used herein refers to a disease characterized by a pathological proliferation of cells or tissues, and subsequent migration or invasion of other tissues or organs. Tumor growth is typically uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicle, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissues or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). The tumors described in the present invention may include, but are not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors such as sarcomas and cancers (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, Rhabdomyosarcoma, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, bronchogenic carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, the "tumor" includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, rectal cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

[0056] II. Implementation Method

[0057] In a first aspect, the present application provides a drug-bifunctional linker compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof:

[0058] MLD (I)

[0059] in,

[0060] M is a bifunctional linker attached to the antibody or antigen-binding fragment thereof;

[0061] L is the linker between the bifunctional linkers M and D;

[0062] D is the cytotoxic drug moiety;

[0063] The M represents a structure shown in the following formula (II):

[0064] A in formula (II) is selected from an alkenyl group, a monocyclic group or a condensed ring group, the monocyclic group is an aryl group or a heteroaryl group, the condensed ring group is a heteroaryl group fused with one selected from an aryl group or a heteroaryl group, and R1 represents a single bond, O, N, S or an alkylene group;

[0065] Said A is optionally substituted by one or more substituents selected from X, wherein X is selected from halogen, halogen substituted or unsubstituted alkyl, -NH-S(O)2-alkenyl or -C 1- C6 heteroalkyl-heterocycloalkyl; the heterocycloalkyl is optionally substituted by one or more halogen-substituted alkyl or -C(O)-halogen-substituted alkyl; wherein the one or more X may correspond to or be different;

[0066] In formula (II), m1 and m2 are each independently selected from integers from 0 to 10, for example, 0, 1, 2, 3, 4, 5 or 6.

[0067] Preferably, said A is optionally substituted by 1, 2 or 3 substituents selected from X.

[0068] Preferably, m1 is 0 or 1, and m2 is 0 or 1.

[0069] In one embodiment, A is selected from a C2-C6 alkenyl group, a monocyclic group, or a condensed ring group, wherein the monocyclic group is a 6- to 12-membered aryl group or a 5- to 12-membered heteroaryl group, wherein the 5- to 12-membered heteroaryl group contains 1-3 nitrogen atoms; wherein the condensed ring group is a ring formed by condensing a 5- to 12-membered heteroaryl group with a 6- to 12-membered aryl group or a 5- to 12-membered heteroaryl group, wherein the 5- to 12-membered heteroaryl group contains 1-3 nitrogen atoms as ring atoms;

[0070] Preferably, A is selected from a monocyclic group or a condensed ring group, wherein the monocyclic group is a 6-membered aromatic ring, a 5-membered heteroaromatic ring or a 6-membered heteroaromatic ring, and the condensed ring group is a 6-membered heteroaryl group fused with a 6-membered aryl group, and the 5-membered heteroaryl group or the 6-membered heteroaryl group contains 1-3 nitrogen atoms;

[0071] Preferably, A is selected from vinyl,

[0072] Preferably, A is selected from vinyl, In one embodiment, X represents halogen, halogen-substituted C1-C6 alkyl, -NH-S(O)2-C2-C6 alkenyl or -C1-C6 heteroalkyl-3 to 8-membered heterocycloalkyl; the 3 to 8-membered heterocycloalkyl is optionally substituted with one or more halogen-substituted C1-C6 alkyl or -C(O)-halogen-substituted C1-C6 alkyl.

[0073] In one embodiment, M is selected from:

[0074] In one embodiment, L is selected from a divalent structure consisting of one or more of the following: Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Gly-Lys, Gly-Phe, Gly-Leu, Gly-Gly-Lys, Gly-Val-Ala, Gly-Val-Cit, Gly-Phe-Gly, Val-Ala-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Leu-Gly, Gly-Gly- Phe-Gly, Gly-Gly-Leu-Gly, Gly-Val-Ala-Gly, Gly-Val-Cit-Gly and Gly-Gly-Gly-Gly-Gly;

[0075] Preferably, L is selected from Val-Cit, Val-Ala, Gly-Gly-Lys, Gly-Phe-Gly, Gly-Lys, Gly-Phe, Gly-Leu, Val-Ala-Gly, Gly-Gly-Phe-Gly, Gly-Val-Ala-Gly, Ala-Ala-Asn and Gly-Leu-Gly;

[0076] Preferably, L is selected from Gly-Lys and Gly-Val-Ala.

[0077] In one embodiment, D is at least one selected from anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors, DNA synthesis inhibitors, RNA polymerase inhibitors, and splicesome inhibitors.

[0078] Preferably, the tubulin inhibitor is an auristatin or maytansine compound; auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division and exhibit anticancer and antifungal activity. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E).

[0079] Preferably, the DNA intercalator is pyrrolobenzodiazepine (PBD).

[0080] Preferably, the non-limiting examples of the DNA topoisomerase inhibitor include topoisomerase I inhibitors or topoisomerase II inhibitors; preferably, the non-limiting examples of the topoisomerase I inhibitor include camptothecin, hydroxycamptothecin, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, SN-38, irinotecan, topotecan, belotecan, ixitecan or rubitecan, Genz-644282; preferably, the non-limiting examples of the topoisomerase II inhibitor include doxorubicin, doxorubicin, oxadiazine ... Preferably, non-limiting examples of the DNA synthesis inhibitor include gemcitabine and other pyrimidine nucleotide analogs; preferably, non-limiting examples of the RNA polymerase inhibitor include α-amanitin; preferably, non-limiting examples of the splicesome inhibitor include thailanstatin series toxins and pharmaceutically acceptable salts, esters and analogs thereof.

[0081] In one embodiment, the L is directly connected to D, or a spacer unit E is connected between the L and the D, and the spacer unit E is selected from one of p-aminobenzyl carbonate amide, p-aminobenzyl carbonate diester, hemiaminal (e.g., aminomethyl ether (also called alkyloxyamine)) and methylenediamine.

[0082] In one embodiment, the M is directly connected to L, or a spacer unit F is connected between the M and the L, and the spacer unit F is -NH-(CH2CH2-O) q -CH2CH2-C(=O)- or a single bond, wherein q represents an integer of 1 to 6, for example, 0, 1, 2, 3, 4, 5 or 6.

[0083] In one embodiment, the compound is selected from the group consisting of:

[0084] Wherein, L and D are defined as above;

[0085] R X1 、R X2 Each is independently selected from halogen or C2-C6 alkenyl;

[0086] Preferably, R X1 is selected from bromine or chlorine, R X2 For vinyl.

[0087] In one embodiment, the compound is selected from the group consisting of:

[0088] In a second aspect, the present application provides a drug-antibody conjugate represented by formula (III) or a pharmaceutically acceptable salt thereof:

[0089] Ab-MLD (III)

[0090] wherein M, L, and D are each defined as a compound of formula (I); Ab is an antibody or an antigen-binding fragment thereof;

[0091] In one embodiment, the drug-antibody conjugate is further linked to other cytotoxic drugs;

[0092] Preferably, the drug-antibody conjugate has a structure represented by formula (IV):

[0093] D'-L'-Ab-MLD (IV)

[0094] Wherein, L' is a linker between the antibody and D', which is the same as or different from L;

[0095] D' is a cytotoxic drug moiety which is the same as or different from D.

[0096] Preferably, D' is exotecan or monomethyl auristatin E. Preferably, the antibody is a monoclonal antibody; preferably, the monoclonal antibody is a human antibody or a humanized antibody; preferably, the monoclonal antibody is IgG; preferably, the monoclonal antibody is IgG1, IgG2, IgG3, or IgG4; preferably, the monoclonal antibody is IgG1 or IgG4 monoclonal antibody; preferably, the IgG1 monoclonal antibody is Herceptin (Trastuzumab (U.S. Patent No. 5,821,337)).

[0097] In one embodiment, Ab is an antibody to a tumor-associated antigen;

[0098] Preferably, the Ab is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.

[0099] Preferably, Ab is Pertuzumab HS627.

[0100] In one embodiment, D or D' is selected from:

[0101] In one embodiment, the present application provides the following drug-antibody conjugates,

[0102] Wherein, L and D are defined the same as those in the compound of formula (I); Ab is an antibody or an antigen-binding fragment thereof.

[0103] n is an integer selected from 1 to 12, preferably an integer selected from 2 to 4; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0104] In a third aspect, the present invention provides the following bifunctional linker compounds:

[0105] R X1 、R X2 Each is independently selected from halogen or C2-C6 alkenyl;

[0106] Preferably, R X1 is selected from bromine or chlorine, R X2 For vinyl.

[0107] The present application provides a method for preparing the drug-antibody conjugate represented by the above formula (III) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0108] The compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is linked to the antibody or antigen-binding fragment thereof.

[0109] The present application provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof or the above-mentioned antibody-drug conjugate and a pharmaceutically acceptable excipient.

[0110] The present application provides the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, the above-mentioned antibody-drug conjugate, and the above-mentioned pharmaceutical composition for use in the preparation of drugs for treating cancer.

[0111] The present application provides a method for treating cancer, comprising the steps of administering the aforementioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, the aforementioned antibody-drug conjugate, or the aforementioned pharmaceutical composition to a patient in need thereof;

[0112] In some embodiments, the cancer comprises gastric cancer, esophageal cancer, cardia cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, primary liver cancer, pancreatic cancer, acute and chronic myeloid leukemia, choriocarcinoma, lung cancer, bladder cancer, intestinal cancer or small cell lung cancer; preferably, the cancer is esophageal cancer, pancreatic cancer or gastric cancer, and further preferably, the cancer is pancreatic cancer.

[0113] Example

[0114] abbreviation:

[0115] Fmoc: 9-fluorenylmethoxycarbonyl; Val: valine, its structural formula is Ala: alanine, its structural formula is Cit: Citrulline, its structural formula is Lys: lysine, its structural formula is Gly: glycine, its structural formula is Phe: phenylalanine, its structural formula is PAB: m: MC: 6-maleimidocaproyl; VC: Val-Cit valine-citrulline; THF: tetrahydrofuran; DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; DMF: N,N-dimethylformamide; DIAD: diisopropyl azodicarboxylate; TFA: trifluoroacetic acid; LAH: lithium aluminum hydride; EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; DBU: 1,8-diazobisspiro[5.4.0]undec-7-ene; TEA: triethylamine; MMAE: monomethyl auristatin E; HOBT: 1-hydroxybenzotriazole; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; DMA: N,N-dimethylacetamide; mCPBA: m-chloroperbenzoic acid; DIC: N,N-diisopropylcarbodiimide; TFA: trifluoroacetic acid; NMP: N-methylpyrrolidone; DMA: N,N-dimethylaniline; NAC: N-acetylcysteine;

[0116] In this embodiment, ADC prepared using but not limited to pertuzumab, pertuzumab (HS627, PERTUZUMAB, purchased from Borri Bio, batch number 0362721003).

[0117] Example 1: Synthesis of 4-((1,2-bis(2-bromoacetyl)pyrazolidin-4-yl)oxy)benzoic acid (CL-01)

[0118] Under ice-bath cooling, chloromethyl oxirane (12 g, 129.70 mmol, 10.14 mL) was slowly added dropwise to a solution of 85% hydrazine hydrate N2H4.H2O (8.12 g, 162.12 mmol, 7.90 mL) in water (10 mL). After the addition, the ice-bath was removed and stirring was continued for 2 hours. Acetone (30 mL) was added and stirring was continued for 2 hours. The non-polar material was extracted with ethyl acetate (3×50 mL). The aqueous phase was separated and potassium carbonate (22.41 g, 162.12 mmol), THF (30 mL) and Boc2O anhydride (35.38 g, 162.12 mmol, 37.21 mL) were added to the aqueous phase under ice-bath cooling. Stirring was continued for 2 hours. The organic phase was extracted with ethyl acetate (100 mL). HCl (50 mL), saturated aqueous NaHCO₃ (100 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the bis-Boc-substituted intermediate CL-01-2 (30 g, 83% yield). CL-01-2 (6 g, 13.82 mmol), THF (40 mL), tert-butyl 4-hydroxybenzoate (2.67 g, 13.76 mmol), and triphenylphosphine (1.69 g, 24.87 mmol) were added to the reaction flask. DIAD (5.04 g, 24.87 mmol, 4.89 mL) was added dropwise to the reaction flask with stirring at 0°C. After the addition, the mixture was stirred at room temperature overnight. Water (50 mL) was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (PE / EA = 4:1) to obtain the intermediate CL-01-3 (8 g, 81.93% yield, HPLC 99%). 1 H NMR(500MHz, CDCl3)δ7.88–7.84(m,2H),6.76-6.72(m,2H),5.01(t,J=4.7Hz,1H),4.23(d,J=12.9Hz,1H), 4.03-3.98(m,1H),3.51(d,J=12.4Hz,1H),3.22(d,J=12.4Hz,1H),1.50(s,9H),1.42(s,9H),1.38(s,9H); 13 CNMR (126MHz, CDCl3) δ165.32,159.82,131.54,125.45,114.73,81.74,81.43,80.76,77.32,77.06,76.81,28.25,28.22,28.05; LCMS: m / z 487.30[M+H] + (Theoretical value 465.56).

[0119] CL-01-3 (8 g, 17.22 mmol), DCM (50 mL) and TFA (15 mL) were added to a reaction flask and stirred at room temperature for 5 hours. The mixture was concentrated and methyl tert-butyl ether was added and stirred to precipitate a white solid product CL-01-4 (3 g, yield 83%, HPLC 99%). 1 H NMR(500MHz,DMSO-d6)δ7.93-7.91(d,J=10Hz,2H),7.07-7.05(d,J=10Hz,2H),5.36(q,J=3.5,1.9Hz, 1H),3.52(d,J=5.2Hz,1H),3.49(d,J=5.1Hz,1H),3.37(d,J=1.6Hz,1H),3.36-3.33(d,J=1.6Hz,1H); 13 C NMR (126MHz, DMSO-d6) δ167.32,160.45,131.92,124.31,115.66,77.38,68.73,52.6; LCMS: m / z 209.61[M+H] + (Theoretical value 209.22).

[0120] CL-01-4 (1.5 g, 7.2 mmol) was added to a reaction flask, followed by THF (50 mL) and solid potassium carbonate (2.99 g, 21.61 mmol). A solution of bromoacetyl bromide (3.64 g, 18.01 mmol, 1.56 mL) in THF (10 mL) was added dropwise with stirring at 0°C. The mixture was stirred at room temperature for 2 hours, then poured into ice water with stirring. The pH was adjusted to acidic with hydrochloric acid, and the white solid product CL-01 (2 g, yield 61%, HPLC 98%) was precipitated by filtration. 1 H NMR (500MHz, DMSO-d6) δ7.91(s,1H),7.89(s,1H),7.02(s,2H),5.46-5.38(m,1H),4.52-4.45(m,2H),4.17-4.14(m,1H),3.51-3.44(m,1H); LCMS: m / z 451.08[M+H] + (Theoretical value 451.08).

[0121] Example 2: Synthesis of 2-((4,6-dichloro-1,3,5-triazine-2-)oxy)acetic acid (CL-02)

[0122] To the reaction flask at 0°C were added DIPEA (3.07 g, 23.73 mmol, 4.13 mL), tert-butyl 2-hydroxyacetate (2.87 g, 21.58 mmol), DCM (50 mL), and a solution of 2,4,6-trichloro-1,3,5-triazine (4 g, 21.57 mmol) in DCM (150 mL). The reaction solution was stirred at 0°C for 1 h, poured into ice water, and the organic phase was separated and washed with saturated sodium chloride solution. The solvent was concentrated and purified by silica gel column chromatography (EA / PE = 2-8% EA) to give tert-butyl 2-((4,6-dichloro-1,3,5-triazine-2-yl)oxy)acetate (4.9 g, 17.43 mmol, yield 80.80%, HPLC 99.9%) as a viscous product, which became a solid upon standing. 1 H NMR (600MHz, CDCl3) δ4.81 (s, 2H), 1.41 (s, 9H); 13 C NMR (151MHz, CDCl3) δ172.59,170.98,165.26,83.55,77.25,77.04,76.82,65.40,27.98.

[0123] Tert-butyl 2-((4,6-dichloro-1,3,5-triazine-2-yl)oxy)acetate (6.5 g, 23.12 mmol) was dissolved in 4N HCl in ethyl acetate and stirred at room temperature overnight. The solvent was concentrated, methyl tert-butyl ether was added, the precipitated solid was filtered, and dried to give the product 2-((4,6-dichloro-1,3,5-triazine-2-yl)oxy)acetic acid CL-02 (3.5 g, 15.24 mmol, yield 65.93%, HPLC 98%); LC / MS m / z 223.38 [M+H] + (Theoretical value 222.96).

[0124] Example 3:

[0125] Synthesis of 4-(2-chloromethyloxirane-2-methoxy)benzoic acid (CL-03)

[0126] To a reaction flask, tert-butyl 4-hydroxybenzoate (0.78 g, 4.00 mmol), 3-chloro-2-chloromethyl-1-propene (998.77 mg, 7.93 mmol), K2CO3 (1.66 g, 12 mmol), and acetonitrile (10 mL) were added. The reaction mixture was heated to 70°C with stirring for 5 h, filtered, and the filtrate was concentrated to dryness. The mixture was purified by silica gel column chromatography (EA / PE = 3-10% EA) to give tert-butyl 4-((2-chloromethyl)allyloxy)benzoate, CL-03-1 (0.6 g, 2.09 mmol, yield 52.39%, HPLC 99%). 1H NMR (600MHz, CDCl3) δ7.90-7.83(m,1H),6.90-6.81(m,1H),5.33(s,1H),5.29(s,1H),4.60(s,1H),4.11(s,1H),1.50(s,5); 13 C NMR (151MHz, CDCl3) δ165.50,161.73,140.30,131.42,124.99,117.81,114.17 ,80.61,77.27,77.06,76.85,67.95,44.97,34.94,31.52,30.15,29.71,28.26.

[0127] To the reaction flask, mCPBA (1.09 g, 6.31 mmol), CL-03-1 (0.6 g, 2.11 mmol) and DCM (10 mL) were added and stirred at room temperature for 36 h. Saturated aqueous sodium sulfite solution (10 mL) was added, and the organic phase was separated and purified by silica gel column chromatography (EA / PE = 5-12% EA) to obtain a white solid product, tert-butyl 4-((2-chloromethyl)oxirane-2-methoxy)benzoate, CL-03-2 (0.42 g, 1.40 mmol, yield 66.00%, HPLC 99.6%).

[0128] 1 HNMR (600MHz, CDCl3) δ8.01-7.76(m,2H),6.96-6.72(m,2H),4.22(dd,J=10.7,1.1Hz,1H),4.11(d,J=10.5Hz,1H),3.79(dt, J=11.8,1.1Hz,1H),3.58(d,J=11.7Hz,1H),2.93(dd,J=4.4,1.0Hz,1H),2.87(dd,J=4.3,1.2Hz,1H),1.52(d,J=13.1Hz,8H); 13 C NMR (151MHz, CDCl3) δ165.41,161.55,131.46,125.35,114.06,80.69,77.28,77.07,76.85,67.47,57.46,51.28,44.81,29.70,28.24LCMS: m / z 298.86[M+H] + (Theory 298.10).

[0129] TFA (1.5 mL), CL-03-2 (0.2 g, 667.18 μmol) and DCM (6 mL) were added to the reaction flask and stirred at room temperature for 2 h. The solvent was removed under reduced pressure to obtain 4-((2-chloromethyl)oxirane-2-methoxy)benzoic acid, CL-03 (0.16 g, 430.64 μmol, yield 64.55%, HPLC 96%). 1 H NMR(600MHz,DMSO-d6)δ7.86-7.79(m,2H),6.99(d,J=8.6Hz,2H),4.23(d,J=11.0Hz,1H) ,4.17(d,J=11.0Hz,1H),3.87-3.79(m,2H),2.96(d,J=4.9Hz,1H),2.92(d,J=4.9Hz,1H); 13 C NMR (151MHz, DMSO) δ167.47,162.07,131.84,124.08,114.85,67.34,57.53,50.50,45.76.LCMS:m / z[M+H] + 243.45 (theoretical value 242.03).

[0130] Example 4: Synthesis of 2-(4,5-dichloromethyl-1-H-1,2,3-triazole-1-)acetic acid (CL-04)

[0131] Tert-butyl bromoacetate (2 g, 10.20 mmol, 1.50 mL) was added dropwise to a reaction flask containing NaN3 (663.06 mg, 10.20 mmol) and DMF (10 mL). The mixture was heated to 70°C and stirred overnight. The mixture was cooled, and water (25 mL) was added. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl azidoacetate (1.2 g, yield 75%) as a light yellow oil. Add tert-butyl azidoacetate CL-04-1 (500 mg, 3.16 mmol), toluene (10 mL) and 1,4-dichloro-2-butyne (777.48 mg, 6.27 mmol) to a reaction flask, heat and stir overnight, concentrate the solvent under reduced pressure, add methyl tert-butyl ether, and stir to precipitate the off-white solid product tert-butyl 2-(4,5-dichloromethyl-1-H-1,2,3-triazole-1-)acetate CL-04-2 (500 mg, yield 56%); 1 H NMR (600MHz, CDCl3) δ5.16(s,2H),4.75(s,2H),4.73(s,2H),1.50(s,9H); LCMS: m / z 280.41[M+H] +(Theoretical value 280.15). 2-(4,5-dichloromethyl-1-H-1,2,3-triazole-1-)acetic acid tert-butyl ester (200 mg, 0.7 mmol), 4.0 M ethyl acetate solution HCl (25.94 mg, 0.7 mmol, 4 mL), and ethyl acetate (5 mL) were added to a reaction flask and stirred at room temperature for 6 hours. The solvent was concentrated under reduced pressure, and methyl tert-butyl ether was added. The mixture was stirred and filtered to precipitate the gray solid product 2-(4,5-dichloromethyl-1-H-1,2,3-triazole-1-)acetic acid CL-04 (120 mg, yield 75%, HPLC 98%). 1 H NMR (600MHz, DMSO-d6) δ5.34(s,2H),5.06(s,2H),4.94(s,2H); LCMS: m / z 224.45[M+H] + (Theoretical value 224.04).

[0132] Example 5: Synthesis of 2-(4,5-dibromomethyl-1-H-1,2,3-triazole-1-)acetic acid (CL-05)

[0133] 2-(4,5-Dibromomethyl-1-H-1,2,3-triazole-1-)acetic acid can be synthesized according to the method for synthesizing 2-(4,5-dichloromethyl-1-H-1,2,3-triazole-1-)acetic acid (CL-04) to obtain a gray solid product, tert-butyl 2-(4,5-dibromomethyl-1-H-1,2,3-triazole-1-)acetate (16 g, yield 58%, HPLC 98%); 1 H NMR (600MHz, CDCl3) δ5.14(s,2H),4.60(s,2H),4.53(s,2H),1.50(s,9H); LCMS: m / z 370.19[M+H] + (Theoretical value 370.06); then treated with trifluoroacetic acid to obtain a white solid product 2-(4,5-dibromomethyl-1-H-1,2,3-triazole-1-)acetic acid, CL-05 (10 g, yield 72%, HPLC 98%); 1 H NMR (600MHz, DMSO-d6) δ5.34(s,2H),5.06(s,2H),4.94(s,2H); LCMS: m / z 314.22[M+H] + (Theoretical value 313.95).

[0134] Example 6: Synthesis of 2-((2,6-dibromomethyl)pyridine-4-oxy)acetic acid (CL-06)

[0135] Dissolve 4-hydroxypyridine-2,6-dicarboxylic acid (5 g, 27.16 mmol) in methanol (50 mL) and add concentrated sulfuric acid. ( The reaction mixture was stirred, heated under reflux for 4 h, cooled to room temperature, poured into ice water, and the pH was adjusted to 5. The precipitated solid product was filtered and recrystallized from ethyl acetate / petroleum ether to give methyl 4-hydroxypyridine-2,6-dicarboxylate (5.76 g, yield 70%). 1 H NMR (600MHz, DMSO-d6) δ11.60(s,1H),7.59(s,2H),3.90(s,6H); LCMS: m / z 212.5[M+H] + (Theoretical value 211.05).

[0136] Methyl 4-hydroxypyridine-2,6-dicarboxylate (1 g, 4.71 mmol) was dissolved in THF (8 mL). A solution of LAH (447.19 mg, 11.78 mmol) in THF (15 mL) was slowly added dropwise under ice-cooling. After the addition, the reaction mixture was stirred at 50°C for 18 h, cooled to room temperature, and quenched by adding saturated aqueous sodium sulfate. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a solid product, CL-06-1 (0.45 g, 72% yield). 1 H NMR(600MHz, DMSO-d6)δ6.16(s,2H),4.24(s,4H); LCMS: m / z 156.65[M+H] + (Theoretical value 155.06).

[0137] To the reaction flask were added tert-butyl 2-bromoacetate (2.62 g, 13.38 mmol, 1.97 mL), CL-06-1 (2.1 g, 13.45 mmol), K2CO3 (2.7 g, 20 mmol) and acetonitrile (25 mL). The reaction solution was heated, stirred and refluxed for 2 h, concentrated, extracted with DCM, and purified by silica gel column chromatography (MeOH / DCM (3-10% MeOH) to give a white solid product, CL-06-2 (1 g, 24% yield); 1 H NMR (500MHz, DMSO-d6) δ6.81 (s, 2H), 5.36 (t, J = 5.8Hz, 2H), 4.74 (s, 2H), 4.46 (d, J = 5.7Hz, 4H), 1.43 (s, 9H); LCMS: m / z 270.86 [M+H] + (Theoretical value 269.13).

[0138] Triphenylphosphine (2.18 g, 8.32 mmol), carbon tetrabromide (2.76 g, 8.32 mmol), CL-06-2 (0.9 g, 3.33 mmol) and DCM (30 mL) were added to the reaction flask and stirred at room temperature for 10 h. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (EtOAc / PE = 0-30% EA) to give the white solid product CL-06-3 (1.06 g, yield 76%). 1 H NMR (500MHz, CDCl3) δ6.88(s,2H),4.59(s,2H),4.47(s,4H),1.50(s,9H); LCMS: m / z394.23[M+H] + (Theoretical value 392.96).

[0139] TFA (2 mL), CL-06-3 (1 g, 2.52 mmol), and DCM (8 mL) were added to the reaction flask. The reaction solution was stirred at room temperature for 15 h, concentrated under reduced pressure, and methyl tert-butyl ether was added to obtain a solid product, 2-((2,6-dibromomethyl)pyridin-4-oxy)acetic acid, CL-06 (620 mg, 53% yield); LCMS: m / z 338.27 [M+H] + (Theoretical value 336.89).

[0140] Example 7: Synthesis of 3,4-bis(ethylenesulfonylamino)benzoic acid (CL-07)

[0141] 3,4-Diaminobenzoic acid (2 g, 13.14 mmol) and methanol (80 mL) were added to the reaction flask and stirred under ice-bath cooling. SOCl2 (3.13 g, 26.29 mmol, 1.92 mL) was added dropwise. After addition, the mixture was stirred at room temperature for 5 h and concentrated under reduced pressure to obtain methyl 3,4-diaminobenzoate. CL-07-1 (2 g, 12.04 mmol, yield 91.56%) was directly used in the next reaction.

[0142] CL-07-1 (300 mg, 1.81 mmol) and DCM (8 mL) were added to the reaction flask and stirred under ice bath. TEA (913.39 mg, 9.03 mmol, 1.26 mL) was then added, followed by the slow dropwise addition of 2-chloroethanesulfonyl chloride (588.61 mg, 3.61 mmol). Stirring was continued at room temperature for 2 h after the addition was complete. The solvent was removed under reduced pressure, and THF and water (3 mL, 1:1) were added. LiOH.H2O (16.96 mg, 404.18 μmol) was added, and the mixture was stirred at room temperature for 1 h. The pH was adjusted to <3 with 2N HCl, and the white solid product CL-07 (22 mg, 62.89 μmol, 31.12% yield, HPLC 95%) was precipitated by filtration. 1 H NMR (500MHz, DMSO-d6) δ13.04(s,1H),9.41(d,J=42.7Hz,2H),7.88(d,J=2.0Hz,1H),7.75(dd,J=8.5,2.0H z,1H),7.47(d,J=8.5Hz,1H),6.92-6.89(m,1H),6.88-6.86(m,1H),6.21-6.11(m,2H),6.10-6.00(m,2H); 13 CNMR (126MHz, DMSO) δ166.75,136.30,135.45,129.41,128.90,128.12,128.07,127.52,126.76,121.67.

[0143] Example 8: Synthesis of 3,5-bis(ethylenesulfonamido)benzoic acid (CL-08)

[0144] CL-08 (20 mg, total yield of three steps 26.80%, HPLC 97%) was successfully obtained according to the above method for preparing CL-07. 1 H NMR (600MHz, DMSO-d6) δ13.10(s,1H),10.32(d,J=6.7Hz,2H),7.42(d,J=6.1Hz,2H),7.28(d,J =6.2Hz,1H),6.77(dt,J=16.9,8.4Hz,2H),6.15(dd,J=16.5,7.2Hz,2H),6.09(t,J=8.4Hz,2H); 13 C NMR (151MHz, DMSO-d6) δ166.88,139.38,136.26,132.96,128.93,115.36,113.73.

[0145] Example 9: Synthesis of 4-chloro-3-(chloromethyl)but-2-enoic acid (CL-09)

[0146] 1,3-Dichloroacetone (678.14 mg, 5.30 mmol), tert-butyl triphenylphosphine acetate (2 g, 5.30 mmol), and DCM (20 mL) were added to a reaction flask. The reaction solution was stirred at room temperature for 12 h. The solvent was removed under reduced pressure and purified by silica gel column chromatography (PE / EA = 100:1) to obtain tert-butyl 4-chloro-3-(chloromethyl)-2-butenoate (1 g, 4.29 mmol, yield 80.96%, HPLC 97%). 1 H NMR (500MHz, CDCl3) δ5.92(s,1H),4.70(s,2H),4.17(s,2H),1.43(s,9H); 13 C NMR (126MHz, CDCl3) δ164.04,147.61,124.21,81.76,45.69,38.02,28.08.

[0147] To the reaction flask, tert-butyl 4-chloro-3-(chloromethyl)-2-butenoate (400 mg, 1.77 mmol), TFA (2 mL) and DCM (3 mL) were added and stirred at room temperature for 2 h. The solvent was removed under reduced pressure and filtered to precipitate the white solid product 4-chloro-3-(chloromethyl)-2-butenoic acid, CL-09 (250 mg, 1.41 mmol, yield 79.80%, HPLC 96%). 1 H NMR (600MHz, CDCl3) δ11.55(s,1H),6.06(s,1H),4.72(s,2H),4.23(s,2H); 13 C NMR (151MHz, CDCl3) δ170.23, 152.23, 121.10, 45.37, 37.86.

[0148] Example 10: Synthesis of 4-bromo-3-(bromomethyl)but-2-enoic acid (CL-10)

[0149] tert-Butyl 4-bromo-3-(bromomethyl)-2-butenoate (1.5 g, 4.67 mmol, yield 88.06%, HPLC 98%) was obtained by a method similar to the above synthesis of tert-butyl 4-chloro-3-(chloromethyl)-2-butenoate; 1 H NMR (500MHz, CDCl3) δ5.89(s,1H),4.63(s,2H),4.08(s,2H),1.43(s,9H); 13C NMR (126MHz, CDCl3) δ164.06,148.37,123.87,81.77,34.02,28.09,25.72.

[0150] The above product was treated with trifluoroacetic acid to give 4-bromo-3-(bromomethyl)-2-butenoic acid, CL-10 (1.0 g, 3.79 mmol, yield 79.49%, HPLC 98%); 1 H NMR (500MHz, CDCl3) δ11.32(s,1H),6.00(s,1H),4.65(s,2H),4.12(s,2H); 13 C NMR (126MHz, CDCl3) δ170.09, 153.05, 120.73, 33.41, 25.20.

[0151] Example 11: Synthesis of 4,5-dibromomethyltriazole-1-acetamideethoxyethoxypropionyl-Gly-Lys-PAB-MMAE (C1)

[0152] (1) Synthesis of Fmoc-GK(Trt)-PAB-PNP

[0153] Fmoc-Lys(Trt) (8.01 g, 12.33 mmol) and (4-aminophenyl)methanol (2.00 g, 12.33 mmol) were dissolved in a mixed solvent of DCM (50 mL) and MeOH (50 mL). The mixture was cooled to 0°C with stirring, and EEDQ (3.05 g, 12.33 mmol) was added. The reaction mixture was stirred at 0°C for 6 h. DBU (0.94 g, 6.16 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 3 h. The reaction solution was directly concentrated at room temperature, then dissolved in DCM (50 mL) and added dropwise to MTBE (500 mL). The precipitated white solid was filtered to obtain Lys(Trt)-PAB (5.2 g, 80% yield, HPLC 95%); LCMS: [M+2] + 495.13 (calculated: 493.27).

[0154] Lys(Trt)-PAB (5.2 g, 10 mmol), Fmoc-Gly (3.42 g, 10.2 mmol) and DCM (50 mL) were added to the reaction flask and stirred until completely dissolved. The mixture was cooled to 0°C and EDC hydrochloride (2.87 g, 15 mmol) was added. The reaction was maintained at 0°C for 3 h. The reaction solution was extracted with a column (PE:EA=1:4) to obtain a white solid product, Fmoc-GK(Trt)-PAB (6.5 g, yield 84%, HPLC 94%). 1H NMR(600MHz,DMSO-d6)δ10.00(s,1H),8.12(d,J=7.5Hz,1H),7.95-7.86(m,2H),7 .74(d,J=7.4Hz,2H),7.68-7.57(m,3H),7.53-7.14(m,20H),5.16(t,J=5.3Hz,1H ),4.47(dd,J=16.1,5.9Hz,3H),4.28(dd,J=24.2,6.7Hz,3H),3.72(s,2H),2.52( d,J=7.4Hz,1H),1.97(s,2H),1.52(ddd,J=109.0,87.5,50.6Hz,6H); LCMS:[M+1] + 773.89 (calculated: 772.36).

[0155] Fmoc-GK(Trt)-PAB (5.4 g, 6.65 mmol) was dissolved in DCM (50 mL), and TEA (2.02 g, 19.95 mmol, 2.78 mL) and bis(4-nitrophenyl) carbonate (6.07 g, 19.95 mmol) were added. The mixture was stirred for 2 h. Silica gel was added directly to the reaction solution and the mixture was passed through a column (DCM:EA=5:1) to obtain a white solid product, Fmoc-GK(Trt)-PAB-PNP (5.5 g, yield 85%, HPLC 97%). 1 H NMR(600MHz,DMSO-d6)δ10.12(s,1H),8.36-8.25(m,2H),8.12(t,J=8.3Hz ,1H),7.88(d,J=7.5Hz,2H),7.72-7.52(m,6H),7.43-6.87(m,21H),4.48-4 .37(m,1H),4.29-4.17(m,3H),3.68(d,J=2.6Hz,2H),3.35-3.16(m,1H),2 .48(t,J=7.9Hz,1H),1.93(d,J=6.9Hz,2H),1.71-1.25(m,6H); LCMS:[M+1] + 937.96 (calculated: 937.37).

[0156] (2) Synthesis of Fmoc-GK(Trt)-PAB-MMAE

[0157] To a 100 mL reaction flask at room temperature, Fmoc-GK(Trt)-PAB-PNP (1.58 g, 1.68 mmol), MMAE (1.15 g, 1.60 mmol), HOBT (244.88 mg, 1.60 mmol), DIPEA (310.01 mg, 2.40 mmol, 417.80 μL) and N-methylpyrrolidone (10 mL) were added in sequence. The reaction was stirred at room temperature for 3 h. 5 g of silica gel powder was added to the reaction solution, and the mixture was purified by column chromatography using PE and EA to obtain a light brown solid Fmoc-GK(Trt)-PAB-MMAE (2.2 g, 1.20 mmol, yield 75.3%, HPLC 93%); LCMS: [M+1] + 1517.58 (calculated value: 1516.93).

[0158] (3) Synthesis of GK(Trt)-PAB-MMAE

[0159] Fmoc-GK(Trt)-PAB-MMAE (3.00 g, 1.98 mmol), DBU (301.43 mg, 1.98 mmol, 296.10 μL) and DMF (9 mL) were added to the reaction flask at room temperature and stirred for 1 h. 5 g of silica gel powder was added for sanding and purified by column chromatography using DCM and MeOH to obtain an off-white solid GK(Trt)-PAB-MMAE (1.8 g, 1.25 mmol, 63.2%, HPLC 94%); LCMS: [M+1] + 1295.35 (calculated: 1294.69).

[0160] (4) Synthesis of Fmoc-PEG2-GK(Trt)-PAB-MMAE

[0161] At room temperature, GK(Trt)-PAB-MMAE (1.8 g, 1.39 mmol), Fmoc-PEG2-COOH (555.35 mg, 1.39 mmol), HATU (528.52 mg, 1.39 mmol), and DIPEA (179.65 mg, 1.39 mmol, 242.11 μL) were added to the reaction flask in sequence. The reaction was stirred at room temperature for 1 h, and DCM (200 mL) was added for extraction. The mixture was washed with saturated sodium chloride solution (100 mL*3), dried over anhydrous magnesium sulfate, and filtered. 5 g of silica gel powder was added to the DCM filtrate for sand making. The mixture was purified by column chromatography using petroleum ether and ethyl acetate to obtain a light yellow solid Fmoc-PEG2-GK(Trt)-PAB-MMAE (2 g, 1.15 mmol, yield 82.41%, HPLC 96%); LCMS: [M+1] +1677.48 (calculated value: 1676.62).

[0162] (5) Synthesis of PEG2-GK(Trt)-PAB-MMAE

[0163] At room temperature, Fmoc-PEG2-GK(Trt)-PAB-MMAE (2 g, 1.19 mmol), DBU (181.66 mg, 1.19 mmol, 178.45 μL) and DMF (6 mL) were added to the reaction flask in sequence. The reaction was stirred at room temperature for 1 h. The reaction solution was reversely prepared using a C18 Spherical 20-35 μm Purification was performed by 100A120g reverse phase column chromatography, using acetonitrile (HPLC grade) and 0.05% TFA aqueous solution as mobile phases B2 and A2, respectively. HPLC preparation method (254 nm and 214 nm wavelength monitoring): 6 mL of the sample DMF solution was injected into the reverse phase column, and the DMF solvent was washed away over 5 min (mobile phase ratio A2:B2 = 95:5). The acetonitrile ratio was then increased to 40% over 80 min (mobile phase ratio from A2:B2 = 95:5 to A2:B2 = 60:40). The acetonitrile ratio was then maintained at 40% and the column was flushed for 60 min to flush out the product. The product was collected and the purity was determined by HPLC (220 nm) and LCMS. The product was lyophilized to obtain a white solid powder PEG2-GK(Trt)-PAB-MMAE (1.3 g, 867.36 μmol, yield 72.69%, HPLC 97%). 11H NMR (500 MHz, DMSO-d6) δ 9.99 (d, J = 7.8 Hz, 1H), 8.13 (t, J = 5.7 Hz, 1H), 8.08 (d, J = 7.7 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.71 - 7.56 (m, 2H), 7.52 (ddd, J = 8.5, 4.4, 2.2 Hz, 0H), 7.45 (s, 1H), 7.35 (dtd, J = 34.2, 19.1, 17.0, 9.3 Hz, 10H), 7.23 - 7.14 (m, 1H), 5.76 (s, 0H), 5.06 (ddd, J = 32.9, 23.4, 13.9 Hz, 1H), 4.47 (dt, J = 29.1, 5.9 Hz, 1H), 4.38 (p, J = 7.7 Hz, 1H), 4.31 - 4.17 (m, 2H), 4.03 (q, J = 7.1 Hz, 1H), 4.00 (s, 1H), 3.74 (d, J = 5.6 Hz, 1H), 3.59 (t, J = 6.5 Hz, 2H), 3.47 (s, 2H), 3.39 (s, 11H), 3.22 (dd, J = 22.4, 11.5 Hz, 4H), 3.16 - 3.09 (m, 2H), 2.98 (s, 1H), 2.91 - 2.81 (m, 2H), 2.54 (s, 1H), 2.45 - 2.34 (m, 2H), 2.27 (dd, J = 16.0, 9.3 Hz, 1H), 2.17 - 2.08 (m, 1H), 1.99 (s, 1H), 1.81 (s, 1H), 1.57 (s, 3H), 1.55 - 1.46 (m, 2H), 1.32 (s, 2H), 1.29 - 1.22 (m, 1H), 1.18 (t, J = 7.1 Hz, 1H), 1.08 - 0.96 (m, 3H), 0.91 - 0.72 (m, 7H), 0.80 (s, 3H); LCMS: [M + 1] + 1454.72 (calculated: 1453.88).

[0164] (6) Synthesis of C1-01

[0165] At room temperature, CL-07, 2-[4,5-bis(bromomethyl)triazol-1-yl]acetic acid (86.10 mg, 275.13 μmol), DCM (5 mL), and DIC (17.36 mg, 137.57 μmol, 21.30 μL) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 5 min. PEG2-GK(Trt)-PAB-MMAE (200 mg, 137.57 μmol) was added and the reaction was continued at room temperature for 2 h. 5 g of silica gel powder was added for sanding. The mixture was purified by column chromatography using DCM and MeOH to obtain a light yellow solid C1-01 (190 mg, yield 72.66%, HPLC 92%); LCMS: [M+1] + 1749.64 (calculated value: 1748.81).

[0166] (7) Synthesis of C1

[0167] C1-01 (190 mg, 108.65 μmol), DCM (5 mL), and TFA (745.00 mg, 6.53 mmol, 500 μL) were added to the reaction flask in sequence at room temperature. The mixture was stirred at room temperature for 1 h, concentrated, dissolved in 3 mL of DMF, and purified by C18 reverse-phase column chromatography using acetonitrile (HPLC grade) and 0.1% TFA aqueous solution as mobile phases B2 and A2, respectively, using the HPLC preparation method (monitoring at 254 nm and 214 nm wavelengths). After lyophilization, C1 was obtained as a white solid powder (110 mg, yield 65.86%, HPLC 98%). 11H NMR (600 MHz, DMSO-d6) δ 10.00 (d, J = 10.7 Hz, 1H), 8.54 (h, J = 6.5 Hz, 1H), 8.17 (dd, J = 17.1, 7.0 Hz, 2H), 8.07 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.61 (dd, J = 17.9, 10.3 Hz, 5H), 7.37 - 7.27 (m, 4H), 7.26 (td, J = 7.6, 2.7 Hz, 2H), 7.17 (q, J = 6.0, 4.9 Hz, 1H), 5.14 (s, 1H), 5.16 - 5.09 (m, 1H), 5.04 (d, J = 13.7 Hz, 1H), 4.94 - 4.80 (m, 3H), 4.49 (p, J = 6.4, 5.8 Hz, 1H), 4.45 - 4.37 (m, 2H), 4.26 (dd, J = 19.0, 10.8 Hz, 1H), 4.01 (dd, J = 15.0, 7.2 Hz, 1H), 4.00 - 3.91 (m, 1H), 3.82 - 3.70 (m, 2H), 3.62 (t, J = 6.4 Hz, 2H), 3.57 (s, 5H), 3.57 (dd, J = 12.1, 5.1 Hz, 1H), 3.48 (s, 6H), 3.44 (q, J = 6.8, 6.3 Hz, 2H), 3.33 - 3.25 (m, 1H), 3.24 (t, J = 9.0 Hz, 4H), 3.19 (d, J = 16.0 Hz, 3H), 3.12 (s, 2H), 2.97 (s, 1H), 2.86 (dd, J = 22.4, 13.2 Hz, 3H), 2.77 (h, J = 6.7 Hz, 2H), 2.41 (t, J = 6.6 Hz, 3H), 2.26 (dd, J = 15.4, 9.2 Hz, 1H), 2.16 - 2.03 (m, 2H), 1.61 (dd, J = 9.3, 4.8 Hz, 1H), 1.55 (qd, J = 13.9, 12.9, 7.1 Hz, 3H), 1,48 (s, 1H), 1.36 (td, J = 16.2, 13.4, 7.1 Hz, 1H), 1.33 - 1.26 (m, 2H), 1.25 - 1.14 (m, 1H), 1.01 (ddd, J = 18.8, 17.1, 6.7 Hz, 6H), 0.93 (s, 2H), 0.86 (ddd, J = 15.7, 13.5, 6.6 Hz, 6H), 0.79 (s, 9H), 0.76 (dt, J = 11.6, 7.4 Hz, 4H); LCMS: [M + 1] + 1507.31 (calculated: 1506.49).

[0168] Example 12: Synthesis of 4,5-dibromomethyltriazole-1-acetamideethoxyethoxypropionyl-Gly-Lys-PAB-(10,11-methylenedioxycamptothecin-7-ethyl)-(N-(S)-tetrahydrofuran-3-)amine (C2)

[0169] (1) Synthesis of 2505

[0170] To a reaction flask, (S)-3-aminotetrahydrofuran (64.31 mg, 0.738 mmol), concentrated hydrochloric acid (0.05 mL, 0.6 mmol), DMSO (1.5 mL) and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were added, stirred and heated to 120-130°C for 1 h. The mixture was cooled to room temperature, and methyl tert-butyl ether was added. The precipitated solid was filtered and purified by silica gel column chromatography to give the product 7-(2-(N-(S)-tetrahydrofuran-3-amino))ethyl-10,11-methylenedioxycamptothecin (18 mg, yield 33%, HPLC 95.9%). 1 H NMR (500MHz, DMSO-d6) δ7.61(d,J=3.4Hz,1H),7.47(d,J=3.9Hz,1H),7.22(d,J =4.2Hz,1H),6.50(s,1H),6.29(d,J=4.0Hz,2H),5.42(d,J=4.0Hz,2H),5.21(d ,J=3.2Hz,2H),3.87-3.58(m,4H),3.21(s,2H),2.85(td,J=11.2,10.7,5.8Hz, 2H),1.90(ddt,J=29.9,10.7,6.2Hz,3H),1.68-1.60(m,1H),1.08-0.76(m,3H); 13 C NMR (126MHz, DMSO-d6) δ173.0,157.3,151.3,150.6,149.7,149.4,147.6,146.9,141.2, 128.6,124.9,118.5,105.9,103.1,100.0,96.4,72.9,72.7,66.9,65.7,58.4,50.4,47.6,32.6,30.7,30.6,8.2; LC-MS:[M+H] + 506.31 (theoretical value 505.18).

[0171] (2) Synthesis of GK(Trt)-PAB-2505

[0172] 2505 (4 g, 7.57 mmol) and Fmoc-GK(Trt)-PAB-PNP (7.27 g, 7.57 mmol) were dissolved in 40 mL of NMP, HOBT (1.74 g, 11.35 mmol) and DIPEA (1.47 g, 11.35 mmol, 1.98 mL) were added, and the mixture was stirred at room temperature for 4 h. The reaction product Fmoc-GK(Trt)-PAB-2505 was detected by HPLC (LCMS: [M+2] + 1303.66; theoretical value 1303.53), piperidine (3.45 g, 40.49 mmol, 4 mL) was added directly to the reaction solution, stirred at room temperature for 0.5 h, 300 mL of methyl tert-butyl ether was added, and the precipitate was filtered; the filter cake was redissolved in 80 mL of DCM, 500 mL of methyl tert-butyl ether was added, and a solid precipitated, which was filtered; the filter cake was dried to obtain GK(Trt)-PAB-2505 (6.5 g, 2.96 mmol, yield 39.12%, HPLC 93%); LCMS: [M+1] + 1081.86 (theoretical value 1081.46).

[0173] (3) Synthesis of Fmoc-PEG2-GK(Trt)-PAB-2505

[0174] Fmoc-PEG2 (2.48 g, 5.88 mmol) was dissolved in DCM (100 mL), and GK(Trt)-PAB-2505 (6.5 g, 5.88 mmol), HATU (3.35 g, 8.82 mmol) and DIPEA (1.14 g, 8.82 mmol, 1.54 mL) were added. The mixture was stirred at room temperature for 2 h, and 20 g of silica gel was added. The mixture was purified by column chromatography to obtain Fmoc-PEG2-GK(Trt)-PAB-2505 (5 g, 2.97 mmol, yield 50.53%, HPLC 95%). 1H NMR(500MHz,DMSO-d6)δ9.99(s,1H),8.13(s,1H),8.07(d,J=7.8Hz,1H),7.8 6(d,J=7.5Hz,2H),7.65(dd,J=18.6,7.9Hz,4H),7.53-7.47(m,2H),7.43-7. 23(m,22H),6.49(s,1H),6.27(s,2H),5.76(s,1H),5.43(s,2H),5.25(s,2H) ,5.14(s,2H),4.58(s,1H),4.36(s,1H),4.27(d,J=6.9Hz,2H),4.18(s,1H), 4.10(s,1H),3.90(s,1H),3.76-3.67(m,3H),3.65-3.54(m,5H),3.46(s,5H) ,3.38(d,J=5.7Hz,4H),3.17(s,3H),3.14-3.09(m,2H),2.69(s,1H),2.37(t ,J=6.4Hz,2H),2.18-2.08(m,1H),1.85(dq,J=14.0,7.3Hz,3H),1.66(s,1H) ,1.53(d,J=9.2Hz,2H),1.36-1.21(m,2H),0.88(t,J=7.3Hz,3H); LCMS:[M+1] + 1462.88 (theoretical value 1462.62).

[0175] (4) Synthesis of PEG2-GK(Trt)-PAB-2505

[0176] Fmoc-PEG2-GK(Trt)-PAB-2505 (5.0 g, 3.36 mmol) was dissolved in DCM (30 mL), and DBU (512.02 mg, 3.36 mmol, 502.97 μL) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was added to 300 mL of methyl tert-butyl ether, and a solid precipitated. The solid product was filtered and redissolved in 50 mL of DCM and added to 500 mL of methyl tert-butyl ether, and a solid precipitated. The solid was filtered and dried to give PEG2-GK(Trt)-PAB-2505 (4.5 g, 2.54 mmol, yield 75.45%, HPLC 93%); LCMS: [M+1] + 1240.90 (theoretical value 1240.55).

[0177] (5) Synthesis of C2

[0178] PEG2-GK-PAB-2505 (100 mg, 80.56 μmol) was dissolved in DCM (2 mL), and 2-[4,5-bis(bromomethyl)triazol-1-yl]acetic acid (25.21 mg, 80.56 μmol) and DIC (12.20 mg, 96.67 μmol, 14.97 μL) were added. The mixture was stirred at room temperature for 1 h. The reaction was detected to be complete (LCMS: [M+2] + To the reaction mixture was added TFA (447.00 mg, 3.92 mmol, 0.3 mL), stirred at room temperature for 2 h, concentrated, dissolved in 2 mL of DMF, and purified by reverse-phase chromatography on a C18 Spherical 20-35 μm 100A 40 g column using acetonitrile (HPLC grade) and 0.1% TFA aqueous solution as mobile phases B2 and A2, respectively, using the HPLC preparation method (monitored at 254 nm and 214 nm wavelengths). After lyophilization, the product C2 (58 mg, 51.7% yield, 93% HPLC) was obtained as a yellow solid powder. 1 H NMR(600MHz,DMSO-d6)δ10.07(s,1H),8.20(d,J=7.6Hz,2H),7.85-7.61(m,9H),7.51(s,1H),7.24(s,1H),6 .29(s,2H),5.43(s,2H),5.27(dd,J=12.2,7.8Hz,3H),5.14(s,3H),4.94(s,1H),4.84(s,1H),4.61(s,1H),4 .60-4.56(m,2H),3.77(d,J=5.1Hz,2H),3.57(ddd,J=31.8,23.9,8.3Hz,12H),2.99-2.92(m,2H),2.81-2.73 (m,2H),2.41(s,2H),1.86(dq,J=13.6,7.2Hz,3H),1.54(d,J=7.8Hz,2H),0.88(t,J=7.2Hz,3H); LCMS:[M+2] + 1293.61 (theoretical value 1291.32).

[0179] Example 13: Synthesis of 4,5-dibromomethyltriazole-1-acetamide ethoxyethoxypropionyl-Gly-Val-Ala-ha-amino(methyl)oxyacetyl exitecan (C3)

[0180] (1) Synthesis of C3-1

[0181] To isotecan mesylate (0.26 g, 0.5 mmol), 2 mL of DMF, HATU (0.19 g, 0.5 mmol), DIPEA (0.129 g.1 mmol) and phthaloyl-Ala-ha-amine (N-methyl)oxyacetic acid (200 mg, 0.59 mmol) were added and reacted at room temperature for 15 h. The mixture was diluted with 200 mL of DCM, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a yellow solid C3-1 (0.3 mg, yield 81%, HPLC purity 92%); LCMS: [M+1] + 753.12 (theoretical value: 752.26).

[0182] (2) Synthesis of C3-2

[0183] C3-1 (0.1 g, 0.13 mmol) was dissolved in 5 ml of MeOH, and hydrazine hydrate (39.9 mg, 0.79 mmol) was added. The tube was sealed and the temperature was raised to 60°C for 2 h. The temperature was then lowered and the reaction solution was poured into 100 mL of saturated brine. The solution was extracted with 200 mL of ethyl acetate and concentrated to obtain the crude product C3-2 (0.07 g, yield 77%, HPLC purity 86%). LCMS: [M+1] + 623.15 (theoretical value: 622.26).

[0184] (3) Synthesis of C3-3

[0185] C3-2 (70 mg, 0.112 mmol) was dissolved in 2 mL of DMF, and DIPEA (29 mg, 0.224 mmol) was added and stirred to dissolve. Fmoc-PEG2-Gly-Val (62.16 mg, 0.112 mmol) and HATU (42.56 mg, 0.112 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 1 h. The reaction solution was directly concentrated and silica gel was added. The mixture was purified by silica gel column chromatography to obtain compound C3-3 (81 mg, yield 62%, HPLC purity 93%); LCMS: [M+1] + 1160.24 (theoretical value: 1159.50).

[0186] (4) Synthesis of C3-4

[0187] In a 10 mL single-necked vial, C3-3 (81 mg, 0.069 mmol) was added and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the reaction was allowed to proceed at room temperature for 1 h. The reaction solution was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, and the supernatant removed and dried to afford C3-4 (32 mg, 49% yield, 95% HPLC purity); LCMS: [M+1] +938.02 (theoretical value: 937.43).

[0188] (5) Synthesis of C3

[0189] 2-(4,5-Dibromomethyl-1-H-1,2,3-triazol-1-yl)acetic acid (129.7 mg, 0.416 mmol) and DIC (52.4 mg, 0.416 mmol) were added to a reaction flask and dissolved in DCM (2.5 mL). After stirring at room temperature for 5 min, the mixture was added to a mixed solution of DCM / DMF (2.5 mL / 1.0 mL) containing C3-4 (300 mg, 0.320 mmol) and reacted at room temperature for 1 hour at 25°C. The reaction solution was added dropwise to 50 mL of tert-butyl methyl ether to precipitate a white solid. The solid was dissolved in DMF (2 mL) and eluted by medium-pressure reverse-phase C18 chromatography (gradient: acetonitrile / 0.1% TFA aqueous solution, 0% to 55%, time 100 min) and lyophilized to obtain a yellow solid C3 (151.0 mg, yield 38.3%, HPLC 97%). 1 H NMR(500MHz,DMSO-d6)δ8.60-8.47(m,1H),8.47-8.35(m,11H),8.33-8.20(m,1H),8.17-8.05(m,1H),8.05-7.92(m,1H),7.8 5-7.70(m,2H),7.30(s,1H),5.65-5.55(m,1H),5.42(s,2H),5.05-5.32(m,4H),4.62-4.42(m,6H),4.33-4.24(m,1H),4.24- 4.01(m,5H),3.80-3.65(m,2H),3.64-3.54(m,2H),3.54-3.38(m,6H),3.32-3.04(m,4H),2.47(s,2H),2.42-2.30(m,5H),2. 24-2.10(m,2H),1.92-1.76(m,3H),1.28-1.18(m,1H),1.17-1.08(m,2H),0.92-0.76(m,5H),0.76-0.62(m,4H); LC-MS:[M+H] + 1232.54 (theoretical value 1232.31).

[0190] Example 14: Synthesis of 2,3-dibromomethylquinoxaline-6-carboxamide ethoxyethoxypropionyl-Gly-Lys-PAB-MMAE (C4)

[0191] (1) Synthesis of C4-01

[0192] At room temperature, 2,3-bis(bromomethyl)quinoxaline-6-carboxylic acid (148.57 mg, 412.70 μmol), DCC (42.58 mg, 206.35 μmol), and DCM (5 mL) were added to the reaction flask in sequence. The reaction was continued at room temperature with stirring for 1 h. After filtration, the mixture was transferred to a 100 mL reaction flask. PEG-GK(Trt)-PAB-MMAE (300 mg, 206.35 μmol), DMF (3 mL), and DIPEA (26.67 mg, 206.35 μmol, 35.94 μL) were added in sequence. The reaction was stirred at room temperature for 1 h. 5 g of silica gel powder was added for sanding. The product was purified by column chromatography using DCM and MeOH to obtain a light yellow solid C4-01 (177 mg, yield 38.21%, HPLC 94%); LCMS: [M+1] + 1796.52 (theoretical value: 1795.87).

[0193] (2) Synthesis of C4

[0194] C4-1 (177 mg, 98.56 μmol), DCM (5 mL), and TFA (745.00 mg, 6.53 mmol, 500 μL) were added to the reaction flask in sequence at room temperature. The mixture was stirred at room temperature for 1 h, concentrated, and dissolved in 3 mL of DMF. The product was purified by C18 reverse-phase column chromatography using acetonitrile (HPLC grade) and 0.1% TFA aqueous solution as mobile phases B2 and A2, respectively, using the HPLC preparation method (monitoring at 254 nm and 214 nm wavelengths). After lyophilization, a white solid powder C4 (29 mg, 17.92 μmol, yield 18.18%, HPLC 96%) was obtained. 1H NMR(600MHz,DMSO-d6)δ9.99(s,1H),8.95(q,J=9.5,7.5Hz,1H),8.64-8.56(m,1H),8.29(dt,J=10.1,7.3Hz,1H),8.1 9-8.15(m,1H),8.13(d,J=8.1Hz,1H),8.07(d,J=7.8Hz,1H),7.89(d,J=8.7Hz,0H),7.61(qd,J=9.2,6.3,4.9Hz,5H), 7.35-7.23(m,4H),7.16(s,1H),5.95(dd,J=14.0,5.1Hz,1H),5.41(s,0H),5.05(dd,J=13.3,3.6Hz,3H),4.73(s,0H) ,4.48(d,J=5.8Hz,1H),4.41(d,J=11.3Hz,2H),4.26(dd,J=19.2,10.8Hz,1H),3.99(s,1H),3.80-3.69(m,2H),3.64- 3.53(m,5H),3.53-3.45(m,4H),3.26-3.19(m,5H),3.17(s,1H),3.11(s,2H),3.07-3.02(m,0H),2.97(s,1H),2.90-2 .80(m,3H),2.77(q,J=6.8Hz,2H),2.44-2.36(m,2H),2.26(dd,J=16.0,9.5Hz,1H),2.11(s,1H),1.99(dq,J=21.7,8. 3,7.8Hz,1H),1.81(s,1H),1.74(s,3H),1.53(q,J=8.4,8.0Hz,2H),1.49(s,3H),1.36(d,J=8.5Hz,1H),1.30(s,3H), 1.24(s,1H),1.06-0.96(m,5H),0.85(tt,J=12.9,6.3Hz,5H),0.79(s,11H),0.76(dt,J=11.7,7.3Hz,3H); LCMS:[M+1] + 1554.32 (theoretical value: 1553.54).

[0195] Example 15: Synthesis of 2,3-dibromomethylquinoxaline-6-carboxamide ethoxyethoxypropionyl-Gly-Lys-PAB-(10,11-methylenedioxycamptothecin-7-ethyl)-(N-(S)-tetrahydrofuran-3-)amine (C5)

[0196] At room temperature (25°C), DCM (2 mL), 2,3-bis(bromomethyl)quinoxaline-6-carboxylic acid (1.12 g, 3.10 mmol), and DCC (320 mg, 1.55 mmol) were added sequentially to a 25 mL single-necked bottle. The mixture was stirred at room temperature for 6 h, filtered, and dried to obtain 2,3-bis(bromomethyl)quinoxaline-6-carboxylic anhydride, which was directly used in the next reaction.

[0197] PEG2-GK(Trt)-PAB-2505 (260 mg, 209.44 μmol) was weighed and dissolved in DCM (5 mL). 2,3-Bis(bromomethyl)quinoxaline-6-carboxylic anhydride (147.03 mg, 209.44 μmol) was added and stirred at room temperature for 1 h (LCMS: [M+2]+1582.26 (theoretical value 1580.43). TFA (745.00 mg, 6.53 mmol, 0.5 mL) was added and stirred at room temperature for 2 h. The reaction solution was concentrated and dissolved in 2 mL of DMF. The reaction mixture was purified by using a C18 Spherical 20-35 μm 100A Purification was performed by reverse phase column chromatography on an 80 g column using acetonitrile (HPLC grade) and 0.1% TFA aqueous solution as mobile phases B2 and A2, respectively, using the HPLC preparation method (monitoring at 254 nm and 214 nm wavelengths). The product C5 was lyophilized to obtain a yellow solid powder (38 mg, 26.88 μmol, 12.83% yield, HPLC 94%). NMR (500MHz, DMSO-d6) δ8.14(dd,J=23.0,15.1Hz,3H),7.64(d,J=8.1Hz,6H),7.41(s,2H),5.95(d,J=13.0Hz, 1H),5.43(s,2H),5.27(s,2H),5.13(s,2H),4.41(s,1H),3.82(d,J=71.8Hz,7H),3.61-3.23(m,21H),2.77(d, J=6.0 Hz, 2H), 2.39 (s, 2H), 2.14 (d, J=8.0 Hz, 1H), 1.92-1.69 (m, 4H), 1.68-1.47 (m, 4H), 0.87 (t, J=7.3 Hz, 3H); LCMS: [M+2]+1340.51 (theoretical value 1338.32).

[0198] Example 16: Synthesis of 2,3-dibromomethylquinoxaline-6-carboxamide ethoxyethoxypropionyl-Gly-Val-Ala-ha-amino(methyl)oxyacetyl exitecan (C6)

[0199] At room temperature, DMF (5 mL), C6-1 (0.94 g, 1 mmol) and 2,3-bis(bromomethyl)quinoxaline-6-carboxylic anhydride (0.7 g, 1 mmol) were added to the reaction flask in sequence. The reaction was stirred at room temperature for 0.5 h. The reaction solution was subjected to reverse preparative chromatography and purified using a C18 Spherical 20-35 μm 100A 80 g reverse phase column chromatography. Acetonitrile (HPLC grade) and 0.05% TFA aqueous solution were used as mobile phases B2 and A2, respectively, using the HPLC preparation method (monitoring at 254 nm and 214 nm wavelengths). The product was lyophilized to obtain a yellow solid powder C6 (456 mg, 0.36 mmol, yield 35%, HPLC 97%). 1 H NMR(500MHz,DMSO-d6)δ8.95(dt,J=10.0,5.4Hz,1H),8.71-8.49(m,2H),8.38-8.21(m,1H) ,8.25(s,1H),8.12(dt,J=8.3,4.5Hz,1H),7.98(d,J=7.2Hz,1H),7.72(dd,J=9.7,6.7Hz,2 H),7.29(d,J=3.8Hz,1H),5.59(p,J=7.6,6.0Hz,1H),5.41(s,2H),5.30-5.20(m,1H),5.15 -5.06(m,1H),4.80(s,8H),4.28(dd,J=13.2,6.6Hz,1H),4.24-4.11(m,2H),4.10(s,3H),4. 08-3.94(m,1H),3.77-3.66(m,2H),3.69-3.59(m,1H),3.59(s,1H),3.59-3.51(m,6H),3.4 8(dt,J=11.5,5.3Hz,4H),3.11(d,J=17.2Hz,1H),2.92(d,J=8.3Hz,1H),2.47(s,3H),2.36( s,4H),2.34(d,J=6.3Hz,1H),2.18(p,J=6.6,5.6Hz,2H),2.07(s,1H),1.98(s,1H),1.93-1 .76(m,4H),1.25-1.11(m,4H),0.91-0.78(m,4H),0.70(dd,J=12.8,6.8Hz,5H); LCMS:[M+2] + 1280.01 (theoretical value: 1277.32).

[0200] Example 17: Synthesis of 2,6-dibromomethylpyridine-4-oxyacetamide ethoxyethoxypropionyl-Gly-Lys-PAB-MMAE (C7)

[0201] (1) Synthesis of C7-1

[0202] At room temperature (25°C), PEG-GK(Trt)-PAB-MMAE (150 mg, 103.17 μmol), CL-06 (69.95 mg, 206.35 μmol), and DIC (13.02 mg, 103.17 μmol, 15.98 μL) were added to a 100 mL single-necked bottle in sequence. The reaction was continued by stirring at room temperature for 1 h. 10 g of silica gel powder was added to make sand, and the mixture was purified by column chromatography using DCM and MeOH to obtain a brown solid product C7-1 (130 mg, yield 70.99%); LCMS: [M+1] + 1775.69 (theoretical value: 1774.84).

[0203] (2) Synthesis of C7

[0204] At room temperature (25°C), C7-1 (130 mg, 73.25 μmol), DCM (5 mL), and TFA (83.52 mg, 732.47 μmol, 56.05 μL) were added to a 100 mL single-necked bottle in sequence. The reaction was stirred at room temperature for 1 h. The product was purified using a C18 Spherical 20-35 μm 100A 120 g reverse phase column with acetonitrile and 0.1% TFA aqueous solution as mobile phases B2 and A2, respectively. HPLC preparation method (254 nm and 214 nm wavelength monitoring) was used to collect the product and lyophilize to obtain a white solid product C7 (54 mg, yield 46.66%, HPLC 97%). 1H NMR(600MHz,DMSO-d6)δ10.02-9.97(m,1H),8.25-8.12(m,3H),8.10-8.04(m,1H),7.61(dd,J =18.2,10.4Hz,7H),7.37-7.23(m,7H),7.17(td,J=11.7,10.4,4.2Hz,1H),7.08(s,1H),5.35 (s,1H),5.04(d,J=13.3Hz,2H),4.61(d,J=10.3Hz,5H),4.49(d,J=6.2Hz,1H),4.41(dt,J=8. 6,4.5Hz,2H),4.26(dd,J=18.8,10.7Hz,1H),4.03-3.93(m,2H),3.81-3.70(m,3H),3.61(t,J= 6.5Hz,3H),3.48(s,5H),3.43(t,J=5.9Hz,2H),3.30-3.16(m,9H),3.12(s,2H),2.97(s,2H), 2.86(dd,J=22.4,13.4Hz,3H),2.77(h,J=6.5Hz,3H),2.40(q,J=8.4,6.4Hz,4H),2.26(t,J=13 .0Hz,1H),2.12(d,J=6.8Hz,2H),1.78-1.75(m,3H),1.75-1.70(m,2H),1.54(p,J=7.2Hz,4H) ,1.36(d,J=7.0Hz,1H),1.33-1.29(m,3H),1.06-0.96(m,7H),0.95-0.72(m,14H); LCMS:[M+1] + 1533.38 (theoretical value: 1532.52).

[0205] Example 18: Synthesis of 2,6-dibromomethylpyridine-4-oxyacetamide ethoxyethoxypropionyl-Gly-Val-Ala-ha-amino(methyl)oxyacetyl exitecan (C8)

[0206] At 25°C, C6-1 (100 mg, 106.61 μmol), DCM (2 mL), and CL-06 (36.14 mg, 106.61 μmol) were added sequentially to a 10 mL single-necked eggplant-shaped flask. The reaction was stirred at room temperature for 1 h. The reaction solution was concentrated and the supernatant was dissolved with 3 mL of DMF. Reverse phase preparative chromatography was performed and purified using a C18 Spherical 20-35 μm 100A 40 g reverse phase column. Acetonitrile (HPLC grade) and 0.1% TFA aqueous solution were used as mobile phases B2 and A2, respectively. HPLC preparation method (254 nm and 214 nm wavelength monitoring) was used. The product was collected and its purity was detected by HPLC (220 nm) and LCMS. It was lyophilized to obtain a yellow solid powder C8 (31 mg, 23.64 μmol, yield 22.17%, HPLC 96%). 1 H NMR(600MHz,DMSO-d6)δ8.51(d,J=8.9Hz,1H),8.23(dt,J=11.3,6.0Hz,2H),8. 11(t,J=5.7Hz,1H),7.99(d,J=7.1Hz,1H),7.81-7.73(m,2H),7.31(s,1H),7.0 9(s,1H),5.61(dt,J=8.9,6.0Hz,1H),5.42(s,2H),5.32-5.08(m,2H),4.61(d, J=11.3Hz,5H),4.29(dd,J=13.2,6.7Hz,1H),4.22-4.11(m,3H),4.07(dd,J=11. 6,5.3Hz,2H),3.72(td,J=16.2,5.6Hz,2H),3.58(t,J=6.5Hz,2H),3.48(qd,J= 5.0,2.5Hz,4H),3.43(t,J=6.0Hz,2H),3.27(q,J=5.9Hz,2H),3.23-2.98(m,1H) ,2.47(s,3H),2.41-2.33(m,5H),2.19(q,J=6.4Hz,2H),1.93-1.79(m,3H),1.1 6-1.09(m,3H),0.87(t,J=7.3Hz,3H),0.71(dd,J=15.6,6.7Hz,5H).LCMS:[M+1] + 1260.30 (theoretical value: 1258.99).

[0207] Example 19: Conjugation of C1 and HS627 Antibodies and Preparation of Dual-Drug ADC

[0208] (1) Preparation of HS627-(C1)2 conjugated drugs

[0209] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (2.86 mg / mL, 0.133 mL) solution and reacted at 0°C for 3 h. Compound C1 (10 mg / mL, 0.2 mL) dissolved in DMA (N,N-dimethylacetamide) was added to the above solution system, mixed, and reacted at 25°C for 4 h. After the reaction was completed, NAC (N-acetyl-L-cysteine) solution (10 mg / mL, 0.11 mL) was added to quench the reaction. Small molecules were removed by ultrafiltration to obtain HS627-(C1). n Drug-antibody conjugates.

[0210] UV-HPLC calculated DAR value: n = 2.1

[0211] (2) Preparation of HS627-(C1)4 conjugated drug

[0212] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. Compound C1 (10 mg / mL, 0.3 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. NAC solution (10 mg / mL, 0.11 mL) was added to quench, and small molecules were removed by ultrafiltration to obtain HS627-(C1). n Conjugated drugs.

[0213] UV-HPLC calculated DAR value: n = 3.9

[0214] (3) Preparation of (C6)4-HS627-(C1)2 Dual-Drug Conjugate

[0215] (3.1) Preparation of C6

[0216] In a 10 mL single-necked bottle, DCM (3 mL), C3-4 (32 mg, 0.034 mmol), bromoacetic acid (9.5 mg, 0.068 mmol), and DIC (8.6 mg, 0.068 mmol) were added sequentially. The reaction was stirred at room temperature for 90 min, concentrated, and passed through a silica gel column (DCM:MeOH=95:5) to obtain a light yellow solid product, compound C6 (22 mg, yield 61%, HPLC 96%). 1H NMR (500MHz, DMSO-d6) δ8.49(d,J=8.9Hz,1H),8.33(t,J=5.7Hz,1H),8.24(t,J=6.2Hz,1H),8.11(t,J=5.8Hz,1H),7.98(d,J=7.1Hz,1H),7.75(d,J=8. 5Hz,1H),7.70(d,J=10.8Hz,1H),7.28(s,1H),5.64-5.54(m,1H),5.40(s,2 H),5.23(d,J=18.9Hz,1H),5.04(d,J=18.9Hz,1H),4.33-3.97(m,6H),3.85 (s,2H),3.70(qd,J=16.5,5.7Hz,3H),3.57(t,J=6.5Hz,4H),3.52-3.37(m, 4H),3.26-3.17(m,4H),3.14-3.06(m,1H),2.47(s,3H),2.41-2.28(m,5H), 2.24-2.11(m,J=6.9,6.0Hz,2H),1.84(dh,J=20.8,7.0Hz,3H),1.14(d,J=7 .1Hz, 3H), 0.85 (t, J=7.3Hz, 3H), 0.70 (dd, J=11.4, 6.8Hz, 6H); LCMS: [M+1] + 1058.24 (theoretical value: 1057.36).

[0217] (3.2) Preparation of (C6)4-HS627-(C1)2 Dual-Drug Conjugate

[0218] The above-mentioned HS627-(C1)2 drug-antibody conjugate (10 mg / mL, 2 mL) was added with TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and reacted at 25°C for 4 h. C6 compound (10 mg / mL, 0.15 mL) dissolved in DMA was added and mixed. The mixture was reacted at 25°C for 12 h. NAC solution (10 mg / mL, 0.08 mL) was added for quenching. Small molecules were removed by ultrafiltration membrane to obtain (C6). m -HS627-(C1) n Drug-antibody conjugates.

[0219] UV-HPLC calculated DAR value: m+n=5.9

[0220] Example 20: Conjugation of C2 and HS627 antibodies and preparation of dual-drug ADC

[0221] (1) Preparation of HS627-(C2)2 conjugated drugs

[0222] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 0°C for 3 h. C2 (10 mg / mL, 0.17 mL) dissolved in DMA was added and mixed. The mixture was reacted at 25°C for 4 h. NAC solution (10 mg / mL, 0.11 mL) was added to quench the mixture. Small molecules were removed by ultrafiltration to obtain HS627-(C2). n Drug-antibody conjugates.

[0223] UV-HPLC calculated DAR value: n = 1.9

[0224] (2) Preparation of HS627-(C2)4 conjugated drugs

[0225] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C2 (10 mg / mL, 0.26 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. NAC solution (10 mg / mL, 0.11 mL) was added to quench, and small molecules were removed by ultrafiltration to obtain HS627-(C2). n Drug-antibody conjugates.

[0226] UV-HPLC calculated DAR value: n = 3.8

[0227] (3) Preparation of (C0)4-HS627-(C2)2 Dual-Drug Conjugate

[0228] Take the above HS627-(C2)2 (10 mg / mL, 2 mL), add TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and react at 25 ° C for 4 h. Add C0 (mc-VC-PABC-MMAE) compound (10 mg / mL, 0.18 mL) dissolved in DMA to the above solution system, mix well, react at 25 ° C for 4 h, add NAC solution (10 mg / mL, 0.08 mL) to quench, and remove small molecules through ultrafiltration membrane to obtain (C0) m -HS627-(C2) n Drug-antibody conjugates.

[0229] Among them, mc-VC-PABC-MMAE has the following structural formula:

[0230] UV-HPLC calculated DAR value: m+n=5.7

[0231] Example 21: Antibody Conjugation of C3 and HS627 and Preparation of Dual-Drug ADC

[0232] (1) Preparation of HS627-(C3)2 conjugated drugs

[0233] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 0°C for 3 h. C3 (10 mg / mL, 0.16 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration to obtain HS627-(C3). n Conjugated drugs.

[0234] UV-HPLC calculated DAR value: n = 2.1

[0235] (2) Preparation of HS627-(C3)4 conjugated drugs

[0236] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C3 (10 mg / mL, 0.25 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. NAC solution (10 mg / mL, 0.11 mL) was added to quench, and small molecules were removed by ultrafiltration to obtain HS627-(C3). n Conjugated drugs.

[0237] UV-HPLC calculated DAR value: n = 3.8

[0238] (3) Preparation of (C0)4-HS627-(C3)2 Dual-drug Conjugate

[0239] The above-mentioned HS627-(C3)2 conjugated drug (10 mg / mL, 2 mL) was added with TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and reacted at 25°C for 4 h. The C0 compound (10 mg / mL, 0.18 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.08 mL). The small molecules were removed by ultrafiltration membrane to obtain (C0) m -HS627-(C3) n Conjugated drugs.

[0240] UV-HPLC calculated DAR value: m+n=5.8

[0241] Example 22: Antibody Conjugation of C4 and HS627 and Preparation of Dual-Drug ADC

[0242] (1) Preparation of HS627-(C4)2 conjugated drugs

[0243] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 0°C for 3 h. C4 compound (10 mg / mL, 0.2 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration to obtain HS627-(C4) n Conjugated drugs.

[0244] UV-HPLC calculated DAR value: n = 1.9

[0245] (2) Preparation of HS627-(C4)4 conjugated drugs

[0246] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C4 (10 mg / mL, 0.32 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration to obtain HS627-(C4). n Conjugated drugs.

[0247] UV-HPLC calculated DAR value: n = 3.7

[0248] (3) Preparation of (C6)4-HS627-(C4)2 Dual-Drug Conjugate

[0249] Take the above HS627-(C4)2 (10 mg / mL, 2 mL), add TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and react at 25 ° C for 4 h, add C6 compound (10 mg / mL, 0.15 mL) dissolved in DMA, mix well, react at 25 ° C for 12 h, add NAC solution (10 mg / mL, 0.08 mL) to quench, and remove small molecules through ultrafiltration membrane to obtain (C6) m -HS627-(C4) n Conjugated drugs.

[0250] UV-HPLC calculated DAR value: m+n=5.7

[0251] Example 23: Conjugation of C5 and HS627 antibodies and preparation of dual-drug ADC

[0252] (1) Preparation of HS627-(C5)2 conjugated drugs

[0253] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 0°C for 3 h. C5 (10 mg / mL, 0.17 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration to obtain HS627-(C5). n Conjugated drugs.

[0254] UV-HPLC calculated DAR value: n = 2.0

[0255] (3) Preparation of HS627-(C5)4 conjugated drugs

[0256] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C5 (10 mg / mL, 0.28 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration to obtain HS627-(C5). n Conjugated drugs.

[0257] ADR value calculated by UV-HPLC: n=3.9

[0258] (4) Preparation of (C0)4-HS627-(C5)2 Dual-drug Conjugate

[0259] Take the above HS627-(C5)2 (10 mg / mL, 2 mL), add TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and react at 25 ° C for 4 h, add C0 compound (10 mg / mL, 0.18 mL) dissolved in DMA, mix well, react at 25 ° C for 4 h, add NAC solution (10 mg / mL, 0.08 mL) to quench, and remove small molecules through ultrafiltration membrane to obtain (C0) m -HS627-(C5) n Conjugated drugs.

[0260] UV-HPLC calculated DAR value: m+n=5.8

[0261] Example 24: Conjugation of C6 and HS627 antibodies and preparation of dual-drug ADC

[0262] (1) Preparation of HS627-(C6)2 conjugated drugs

[0263] HS627 antibody (10 mg / mL, 5 mL) was added to TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 0°C for 3 h. C6 (10 mg / mL, 0.16 mL) dissolved in DMA was added to the above solution system, mixed, reacted at 25°C for 4 h, and quenched by adding NAC aqueous solution (10 mg / mL, 0.11 mL). Small molecules were removed by ultrafiltration membrane to finally obtain HS627-(C6). n Conjugated drugs.

[0264] UV-HPLC calculated DAR value: n = 1.9

[0265] (2) Preparation of HS627-(C6)4 conjugated drugs

[0266] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C6 (10 mg / mL, 0.27 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. NAC aqueous solution (10 mg / mL, 0.11 mL) was added to quench, and small molecules were removed by ultrafiltration to obtain HS627-(C6). n Conjugated drugs.

[0267] UV-HPLC calculated DAR value: n = 3.8

[0268] (3) Preparation of (C0)4-HS627-(C6)2 Dual-Drug Conjugate

[0269] Take the above HS627-(C6)2 (10 mg / mL, 2 mL), add TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and react at 25 ° C for 4 h. Add C0 compound (10 mg / mL, 0.18 mL) dissolved in DMA to the above solution system, mix well, react at 25 ° C for 4 h, add NAC solution (10 mg / mL, 0.08 mL) to quench, and remove small molecules through ultrafiltration membrane to obtain (C0) m -HS627-(C6) n Conjugated drugs.

[0270] UV-HPLC calculated DAR value: m+n=5.9

[0271] Example 25: Antibody Conjugation of C7 and HS627 and Preparation of Dual-Drug ADC

[0272] (1) Preparation of HS627-(C7)2 conjugated drugs

[0273] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 10°C for 3 h. C7 (10 mg / mL, 0.20 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. After the reaction was completed, NAC solution (10 mg / mL, 0.11 mL) was added to quench the reaction, and then small molecules were removed through an ultrafiltration membrane to obtain HS627-(C7). n Conjugated drugs:

[0274] The average DAR value calculated by UV-HPLC was: n=2.0.

[0275] (2) Preparation of HS627-(C7)4 conjugated drugs

[0276] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C7 compound (10 mg / mL, 0.33 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. After the reaction was completed, NAC solution (10 mg / mL, 0.11 mL) was added to quench the reaction, and then small molecules were removed through an ultrafiltration membrane to finally obtain HS627-(C7). n Conjugated drugs:

[0277] Average DAR value calculated by UV-HPLC: n=4.1

[0278] (3) Preparation of (C6)4-HS627-(C7)2 conjugated drugs

[0279] The above-mentioned HS627-(C7)2 conjugated drug (10 mg / mL, 2 mL) was added with TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and reacted at 25 ° C for 4 h. The C6 compound (10 mg / mL, 0.15 mL) dissolved in DMA was added and mixed. The reaction was continued at 25 ° C for 12 h. NAC solution (10 mg / mL, 0.08 mL) was added for quenching. The small molecules were removed by ultrafiltration membrane to obtain DAR (2 + 4) product (C6). m -HS627-(C7) nConjugated drugs:

[0280] UV-HPLC calculated average DAR value: m+n=5.9

[0281] Example 26: Antibody Conjugation of C8 and HS627 and Preparation of Dual-Drug ADC

[0282] (1) Preparation of HS627-(C8)2 conjugated drugs

[0283] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.133 mL) and reacted at 10°C for 3 h. C8 (10 mg / mL, 0.16 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. After the reaction was completed, NAC solution (10 mg / mL, 0.11 mL) was added to quench the reaction, and small molecules were removed by ultrafiltration membrane to finally obtain HS627-(C8). n Conjugated drugs:

[0284] Average DAR value calculated by UV-HPLC: n=1.9

[0285] (2) Preparation of HS627-(C8)4 conjugated drugs

[0286] HS627 antibody (10 mg / mL, 5 mL) was added to a TCEP.HCl solution (2.86 mg / mL, 0.2 mL) and reacted at 25°C for 4 h. C8 (10 mg / mL, 0.27 mL) dissolved in DMA was added to the above solution system, mixed, and reacted at 25°C for 4 h. After the reaction was completed, NAC solution (10 mg / mL, 0.11 mL) was added to quench the reaction, and then small molecules were removed through an ultrafiltration membrane to finally obtain HS627-(C8). n Conjugated drugs:

[0287] Average DAR value calculated by UV-HPLC: n=4.1

[0288] (3) Preparation of (C0)4-HS627-(C8)2 conjugated drugs

[0289] Take the above HS627-(C8)2 conjugate drug (10 mg / mL, 2 mL), add TCEP.HCl solution (2.86 mg / mL, 0.08 mL) and react at 25°C for 4 h. Add C0 (MC-VC-PAB-MMAE) compound (10 mg / mL, 0.18 mL) dissolved in DMA to the above solution system, mix well, and react at 25°C for 4 h. After the reaction is completed, NAC solution (10 mg / mL, 0.08 mL) is added to quench, and then small molecules are removed by ultrafiltration membrane bag to finally obtain (C0) m -HS627-(C8) n Conjugated drugs:

[0290] Average DAR value calculated by UV-HPLC: m+n=6.1

[0291] Effect Example 1: DAR value of ADC

[0292] The DAR values ​​of the ADCs prepared in the examples are shown in Table 1 below.

[0293] Table 1 DAR determination results of ADC compounds involved in the embodiments of the present invention

[0294] Effect Example 2: In vivo tumor growth inhibition activity of ADC

[0295] ADC in vitro inhibitory activity test method:

[0296] Human pancreatic cancer Her2 low-expressing CFPAC-1 cells and breast cancer MCE-7 cells were cultured in monolayer in vitro. When the cell saturation reached 80%-90%, they were digested with trypsin-EDTA, centrifuged and the supernatant was discarded. The cells were resuspended in PBS and the cell suspension was adjusted to an appropriate concentration. The cells (2-10×10 6 cells / 0.1 mL) were subcutaneously inoculated into BALB / c nude mice, and the animals and the growth of the transplanted tumors were observed regularly. 3 The mice were randomly divided into two groups according to tumor volume and body weight: a vehicle control group (or a vehicle group, injected with normal saline) and an ADC-treated group (dissolved in normal saline), with 6 animals in each group. The drug was administered intravenously every 7 days for 2 times (the first dose was recorded as Day 1, and the second dose was recorded on Day 7, with a dose of 2 mg / kg or 5 mg / kg). The long diameter a (mm), short diameter b (mm) of the tumor and the body weight of the mice were measured with a vernier caliper twice a week, and the tumor volume (V) was calculated according to the following formula: V = 1 / 2 × a × b 2 (mm 3), where a and b represent tumor length and width, respectively. A growth curve was plotted, and the tumors were removed and weighed. Statistical analysis was performed using GraphPad Prism software based on the data of tumor volume and weight of tumor-bearing mice at the end of the experiment to obtain the tumor inhibition results (Figures 1, 2, 3, and 4). HS627-Dxd was used as a control. Its structural formula is as follows:

[0297] n=8; Main evaluation indicators:

[0298] Tumor growth inhibition (TGI):

[0299] TGI (%) = [1-(avT i-0 / avC i-0 )]×100%; where avT i-0 is the average tumor volume of the dosing group on a specific day, minus the average tumor volume of the dosing group on the day dosing was started; where avCi-0 is the average tumor volume of the vehicle control group on a specific day, minus the average tumor volume of the vehicle control group on the day dosing was started.

[0300] Table 2 Grouping and drug administration settings

[0301] Tumor inhibition rate: The CFPAC-1 model was calculated based on the tumor volume at day 42; the MCF-7 model was calculated based on the tumor volume at day 35.

[0302] As can be seen from the results, the exemplary ADC compounds of the present invention, Entry 2 (DAR2), Entry 5 (DAR4), Entry 8 (DAR4), Entry 17 (DAR4), Entry 18 (DAR2+4), and Entry 24 (DAR2+4), all showed excellent tumor inhibitory effects in pancreatic cancer and breast cancer tumor models.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof: MLD (I) in, M is a bifunctional linker attached to the antibody or antigen-binding fragment thereof; L is a linker between the bifunctional linkers M and D; D is the cytotoxic drug moiety; The M represents a structure shown in the following formula (II): A in formula (II) is selected from an alkenyl group, a monocyclic group or a condensed ring group, the monocyclic group is an aryl group or a heteroaryl group, the condensed ring group is a heteroaryl group condensed with one selected from an aryl group and a heteroaryl group, and R1 represents a single bond, O, N, S or an alkylene group; A is optionally substituted by one or more substituents selected from X, X is selected from halogen, halogen-substituted or unsubstituted alkyl, -NH-S(O)2-alkenyl or -C1-C6 heteroalkyl-heterocycloalkyl; the heterocycloalkyl is optionally substituted by one or more halogen-substituted alkyl or -C(O)-halogen-substituted alkyl; In formula (II), m1 and m2 are each independently selected from integers from 0 to 10; Preferably, A is selected from C2-C6 alkenyl, monocyclic group or condensed ring group, the monocyclic group is a 6- to 12-membered aryl group or a 5- to 12-membered heteroaryl group, the 5- to 12-membered heteroaryl group contains 1-3 nitrogen atoms; the condensed ring group is a ring formed by condensing a 5- to 12-membered heteroaryl group with a 6- to 12-membered aryl group or a 5- to 12-membered heteroaryl group, the 5- to 12-membered heteroaryl group contains 1-3 nitrogen atoms as ring atoms; Preferably, A is selected from a monocyclic group or a condensed ring group, the monocyclic group is a 6-membered aromatic ring, a 5-membered heteroaromatic ring or a 6-membered heteroaromatic ring, the condensed ring group is a 6-membered heteroaryl group fused with a 6-membered aryl group, and the 5-membered heteroaryl group or the 6-membered heteroaryl group contains 1-3 nitrogen atoms; Preferably, A is selected from vinyl, Preferably, X represents halogen, halogen-substituted C1-C6 alkyl, -NH-S(O)2-C2-C6 alkenyl or -C 1- C6 heteroalkyl-3 to 8 membered heterocycloalkyl; the 3 to 8 membered heterocycloalkyl is optionally substituted by one or more halogens. 1- C6 alkyl or -C(O)-halogen substituted C 1- C6 alkyl substitution; Preferably, M is selected from: Preferably, L is selected from a divalent structure consisting of one or more of the following: Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Gly-Lys, Gly-Phe, Gly-Leu, Gly-Gly-Lys, Gly-Val-Ala, Gly-Val-Cit, Gly-Phe-Gly, Val-Ala-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Leu-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Leu- Gly, Gly-Val-Ala-Gly, Gly-Val-Cit-Gly and Gly-Gly-Gly-Gly-Gly; Preferably, L is selected from Val-Cit, Val-Ala, Gly-Gly-Lys, Gly-Phe-Gly, Gly-Lys, Gly-Phe, Gly-Leu, Val-Ala-Gly, Gly-Gly-Phe-Gly, Gly-Val-Ala-Gly, Ala-Ala-Asn and Gly-Leu-Gly; Preferably, L is selected from Gly-Lys and Gly-Val-Ala.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: D is at least one selected from anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors, DNA synthesis inhibitors, RNA polymerase inhibitors, and Splicesome inhibitors, preferably camptothecin or a camptothecin derivative, pyrrolobenzodiazepine (PBD) dimer, methyl auristatin E, or methyl auristatin F.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: The L is directly connected to D, or, A spacer unit E is connected between the L and the D, and the spacer unit E is selected from p-aminobenzyl carbonate amide, p-aminobenzyl carbonate diester, aminomethyl ether and methylenediamine.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: The M is directly connected to L, or, A spacer unit F is connected between the M and the L, and the spacer unit F is selected from -NH-(CH2CH2-O) q -CH2CH2-C(=O)-, wherein q represents an integer of 1-6.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: The compound is selected from the group consisting of: Wherein, L and D are defined as above; R X1 , R X2 Each is independently selected from halogen or C2-C6 alkenyl; Preferably, R X1 is selected from bromine or chlorine, R X2 For vinyl; Preferably, the compound is selected from the group consisting of:

6. The drug-antibody conjugate represented by formula (III) or a pharmaceutically acceptable salt thereof: Ab-MLD (III) in, M, L, and D are each defined as the compound of formula (I); Ab is an antibody or an antigen-binding fragment thereof; Preferably, the Ab antibody is a tumor-associated antigen antibody; Preferably, Ab is an anti-Her2 antibody; Preferably, Ab is Pertuzumab; Preferably, the drug-antibody conjugate compound is also connected to other cytotoxic drugs; Preferably, the drug-antibody conjugate compound has a structure shown in formula (IV): D'-L'-Ab-MLD (IV) Wherein, L' is a linker between the antibody and D', which is the same as or different from L; D' is a cytotoxic drug moiety which is the same as or different from D; Preferably, the drug-antibody conjugate represented by formula (III) or a pharmaceutically acceptable salt thereof is the following drug-antibody conjugate compound, Wherein, L and D are defined as the compound of formula (I); Ab is an antibody or an antigen-binding fragment thereof; n is an integer selected from 1 to 12, preferably an integer selected from 2 to 4; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

7. The following bifunctional linker compounds: in, R X1 , R X2 Each is independently selected from halogen or C2-C6 alkenyl; Preferably, R X1 is selected from bromine or chlorine, R X2 For vinyl.

8. A method for preparing the drug-antibody conjugate compound of formula (III) or a pharmaceutically acceptable salt thereof according to claim 6, comprising the following steps: The compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is linked to the antibody or antigen-binding fragment thereof.

9. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof or the antibody-drug conjugate according to claim 6 and a pharmaceutically acceptable excipient.

10. Use of the compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt, stereoisomer or prodrug, the drug-antibody conjugate according to claim 6, the bifunctional linker compound according to claim 7 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating cancer; Preferably, the cancer includes gastric cancer, esophageal cancer, cardia cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, primary liver cancer, pancreatic cancer, acute and chronic myeloid leukemia, choriocarcinoma, lung cancer, bladder cancer, intestinal cancer or small cell lung cancer; more preferably, the cancer is esophageal cancer, pancreatic cancer or gastric cancer, and further preferably, the cancer is pancreatic cancer.

11. A method for treating cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, a drug-antibody conjugate according to claim 6, a bifunctional linker compound according to claim 7, or a pharmaceutical composition according to claim 9; Preferably, the cancer includes gastric cancer, esophageal cancer, cardia cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, primary liver cancer, pancreatic cancer, acute and chronic myeloid leukemia, choriocarcinoma, lung cancer, bladder cancer, intestinal cancer or small cell lung cancer; more preferably, the cancer is esophageal cancer, pancreatic cancer or gastric cancer, and further preferably, the cancer is pancreatic cancer.

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