Eribulin drug connexon and antibody coupling drug and application thereof

Abstract

The invention provides an eribulin derivative with an anti-tumor effect, an antibody-drug conjugate of the eribulin derivative, and a pharmaceutical composition containing the antibody-drug conjugate.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to eribulin derivatives with anti-tumor effects and antibody-drug conjugates thereof. Background Art

[0002] Small molecule cytotoxins are the most commonly used chemotherapeutic drugs due to their high cytotoxicity and relatively low production costs. However, they have low targeting to cancer cells and rapid plasma clearance. In contrast, modern mAb therapies can target cancer cells with high selectivity by recognizing and binding to cell surface antigens that are overexpressed in certain cancer indications. In addition, mAbs can also show long half-lives and low clearance rates. Antibody-drug conjugates (ADCs) are a rapidly growing class of targeted drug delivery therapeutics, including monoclonal antibodies (mAbs) linked to small molecule cytotoxic payloads via covalent linkers, combining the powerful killing effects of traditional small molecule chemotherapy with the tumor targeting of antibody drugs. ADC drugs are used to treat cancer, and their targeting of the disease is determined by the target targeted by the antibody, and they can perform targeted DNA damage or inhibit microtubules in tumor cells that highly express the target. Due to its high targeting and lethality, it can use highly toxic drugs (IC50:10 -9 mol / L to<10 -10 mol / L)(doi:10.1158 / 1535-7163.MCT-17-1215). ADC drugs have a larger therapeutic safety window than chemotherapy drugs and are relatively safer. As of 2023, there are as many as 15 ADC drugs approved for marketing worldwide, with targets including CD19, CD22, CD30, CD33, CD79b, HER2, Nectin-4, Trop-2, EGFR, Tissue factor and FRα.

[0003] Eribulin is derived from the complex anti-tumor natural product Halichondrin B. In November 2010, the drug was launched in the United States and officially entered the domestic market in 2019 for the treatment of metastatic breast cancer and liposarcoma. Eribulin is a targeted microtubule inhibitor that can inhibit the proliferation of cancer cells by binding to tubulin and microtubules. Due to its high targeting and lethality, Eribulin has become one of the candidate cytotoxins for ADC drugs. In current clinical studies, Eisai's MORAb-202 is the first ADC molecule of Eribulin, an ADC drug targeting folate receptor α (FRα). Phase I clinical trials have been completed and phase II clinical trials are underway. The ADC drug uses farletuzumab (FRα humanized IgG1 monoclonal antibody) as an antibody, -MC-PEG2-VC- as a linker, and Eribulin as a cytotoxin, with a drug-antibody coupling ratio (DAR) of 4.0. This ADC drug can not only rely on antibodies to recognize target antigens, undergo cell internalization, and release cytotoxins to kill tumor cells through lysosomal lysis, but also kill tumor cells through ADCC, ADCP, and CDC effects. In addition, MORAb-202 not only exhibits strong efficacy and targeting, but also exhibits a strong "bystander effect". However, when investigating the effect of retention time in human or mouse plasma on the DAR value of MORAb-202, the researchers found that after 240 hours, the DAR dropped to about 2.6 (doi:10.1158 / 1535-7163.MCT-17-1215). Therefore, it is of far-reaching significance to further develop antibody conjugates derived from eribulin with better plasma stability and safety. At the same time, different biopharmaceutical companies are also committed to studying eribulin-type ADC drugs. In 2021, Hengrui Medicine constructed a new type of Eribulin linker-drug using an etherified derivative of Eribulin as a payload, and conducted in vitro and in vivo experiments to evaluate its efficacy (CN115845080A). In 2023, Kunshan Xinyunda realized the construction of a new type of Eribulin linker-drug with a maleimide linker without self-cleavage fragments (WO2023024949A1), a new type of Eribulin linker-drug with a pyrimidine methyl sulfone linker (WO2023016488A1), and a new type of Eribulin linker-drug with other aromatic heterocyclic methyl sulfone linkers (WO2023124963A1). In 2023, Yilian Bio used a pyrimidine methyl sulfone linker instead of a maleimide linker and a new tumor microenvironment cleavage tripeptide instead of Val-Cit to realize the construction of a new type of Eribulin ADC drug (WO2023155808A1).Existing eribulin-type linker-drugs all produce activity by directly releasing eribulin or its derivatives. The present invention creatively designs an unstable intermediate of eribulin as a payload and connects it to an antibody through a stable linker. The obtained ADC first releases the eribulin derivative after cell internalization, and then the derivative is further converted into eribulin. Summary of the invention

[0004] The present invention provides an antibody-drug conjugate represented by formula (I), and its mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances:

[0005]

[0006]

[0007] Wherein, Ab is an antibody or an antigen-binding fragment thereof;

[0008] m is an integer or decimal selected from 1 to 12 (preferably an integer or decimal of 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8); L is a linker moiety;

[0009] R 1 , R 2 Each is independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxy, optionally substituted with the following substituents: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, C6-C 14 Aryl, 5-14 membered heteroaryl, or R 1 and R 2 Together with the carbon atom to which it is connected, it forms the following optionally substituted substituents: C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, wherein the optionally substituted substituents are selected from one or more of halogen, halogenated C1-C3 alkyl, amino, cyano, nitro, hydroxyl, and hydroxy C1-C3 alkyl.

[0010] In some embodiments, Ab is selected from antibodies or antigen-binding fragments thereof targeting FRα, HER2, HER3, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD73, CD105, EGFR, VEGFR, VEDF, PD-1, PD-L1, MET, RET, B7H3, B7H4, D113.

[0011] In some embodiments, the Ab is selected from an antibody or an antigen-binding fragment thereof that targets FRα.

[0012] In some embodiments, Ab is selected from Farletuzumab, Trastuzumab, Pertuzumab, Nimotuzumab, Emibetuzumab, Besponsa, Pinatuzumab, Brentuximab, Gemtuzumab, Bevacizumab, cBR96, and Glematumamab, or an antigen-binding fragment thereof.

[0013] In some embodiments, Ab is selected from Farletuzumab or an antigen-binding fragment thereof.

[0014] In some embodiments, R 1 , R 2 Each is independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxyl, optionally substituted with the following substituents: C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10 Aryl, 5-10 membered heteroaryl, or R 1 and R 2 Together with the carbon atom to which it is connected, it forms the following optionally substituted substituents: C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, wherein the optionally substituted substituents are selected from one or more of halogen, halogenated C1-C3 alkyl, amino, cyano, nitro, hydroxyl, and hydroxy C1-C3 alkyl.

[0015] In some embodiments, R 1 Selected from hydrogen, R 2 is selected from hydrogen, halogen, amino, cyano, nitro, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, fluorinated C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 Aryl, or R 1 and R 2 Together with the carbon atom to which it is attached, it forms a C3-C6 cycloalkyl group.

[0016] In some embodiments, R 1 Selected from methyl, R 2 is selected from halogen, amino, cyano, nitro, hydroxy, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, fluorinated C1-C3 alkyl, C3-C6 cycloalkyl, C6-C 10 Aryl, or R 1 and R 2Together with the carbon atom to which it is attached, it forms a C3-C6 cycloalkyl group.

[0017] In some embodiments, R 1 , R 2 each independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxy, an optionally substituted substituent selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, methylthio, ethylthio, n-propylthio, isopropylthio, cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, aziridine, azetidinyl, tetrahydropyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, phenyl, naphthyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, or R 1 and R 2 Together with the carbon atom to which it is attached, it forms the following optionally substituted substituents: cyclopropane, cyclobutane, cyclopentane, cyclohexane, aziridine, azetidinyl, tetrahydropyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, wherein the optionally substituted substituents are selected from one or more of fluorine, chlorine, bromine, iodine, monofluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, amino, cyano, nitro, hydroxyl, hydroxymethyl, hydroxyethyl.

[0018] In some embodiments, R 1 , R 2 Each is independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, cyclopropane, cyclobutane, cyclopentane, cyclohexane, phenyl, or R 1 and R 2 Together with the carbon atoms to which it is attached, it forms cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0019] In some embodiments, R 1 Selected from hydrogen, R 2 is selected from hydrogen, fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, cyclopropane, cyclobutane, cyclopentane, cyclohexane, phenyl; or R 1 Selected from methyl, R 2 is selected from fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, cyclopropane, cyclobutane, cyclopentane, cyclohexane, phenyl; or R 1 and R 2 Together with the carbon atoms to which it is attached, it forms cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0020] In some embodiments, R 1, R 2 are selected from hydrogen.

[0021] In some embodiments, R 1 , R 2 are selected from methyl.

[0022] In some embodiments, R 1 Selected from H, R 2 Selected from methyl.

[0023] In some embodiments, R 1 is selected from H or methyl, R 2 Selected from phenyl.

[0024] In some embodiments, R 1 is selected from H or methyl, R 2 Selected from cyclopentyl.

[0025] In some embodiments, R 1 and R 2 Together with the carbon atoms to which it is attached, it forms cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0026] In some embodiments, the linker L is selected from a cleavable linker or a non-cleavable linker.

[0027] In some embodiments, the linker L is -L 1 -L 2 -L 3 -L 4 -L 5 -;

[0028] Where -L 1 -Selected from:

[0029]

[0030] The a-end is covalently linked to the antibody part Ab, and the b-end is covalently linked to L 2 Covalently linked; R 3 selected from hydrogen, halogen, amino, cyano, nitro, hydroxyl, optionally substituted with the following substituents: C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10 Aryl, 5-10 membered heteroaryl.

[0031] In some embodiments, R 3is selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, cyano, nitro, hydroxyl, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, methylthio, ethylthio, n-propylthio, isopropylthio, cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, phenyl, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidine 1,2-b]pyridinyl, 1,2-a]pyridinyl, 1,5-a]pyridinyl, 1,5-a]pyridinyl, 1,2-b]pyridaz ...

[0032] -L 2 - is selected from a bond or a C1-C8 alkylene group, a C3-C8 cycloalkylene group, a 1-100-membered straight-chain heteroalkylene group, or a 3-8-membered heterocyclylene group, wherein the alkylene group, the cycloalkylene group, the heteroalkylene group, and the heterocyclylene group are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, NH2-C1-C6 alkyl, a 1-6-membered heteroalkyl group, a C1-C6 alkoxy group, a halogen, a hydroxyl group, an amino group, a nitro group, a cyano group, a carboxyl group, or a C3-C8 cycloalkyl group, and the heteroatoms in the heteroalkylene group and the heterocyclylene group are selected from one or more of N, O, or S.

[0033] In some embodiments, -L 2 - is selected from a bond, a C1-C8 alkylene, -(CH2CH2O)nCH2-, -(CH2CH2O)nCH2CH2-, -(CH2CH2O)nCH2CH2CH2-, wherein n is selected from an integer of 1-24 (e.g., an integer of 1-12, an integer of 3-8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24);

[0034] In some embodiments, -L 2- is selected from a bond, -CH2CH2CH2CH2CH2-, -(CH2CH2O)nCH2CH2-, n is selected from an integer of 1-24 (such as an integer of 1-12, an integer of 3-8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24);

[0035] -L 3 - is selected from -C(O)-, -S(O)-, -S(O)O-;

[0036] In some embodiments, -L 3 - is selected from -C(O)-;

[0037] -L 4 - a peptide residue selected from 2 to 8 amino acids, wherein the amino acids are selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), cysteine ​​(Cys), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), citrulline (Cit), arginine (Arg) and histidine (His), wherein the nitrogen end is connected to L 3 ;

[0038] In some embodiments, -L 4 - is selected from valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-citrulline (Glu-Val-Cit), alanine-alanine-aspartic acid (Val-Val-Asn), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), wherein the nitrogen end is connected to L 3 ;

[0039] In some embodiments, -L 4 -Selected from:

[0040]

[0041] -L 5 -Selected from:

[0042]

[0043] The nitrogen end is connected to L 4, R is selected from hydrogen, C1-C8 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, halogen, an optionally substituted hydrophilic side chain, wherein the hydrophilic side chain comprises -(CH2CH2O) p -, wherein p and k are each independently selected from an integer of 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

[0044] In some embodiments, R is selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C3-C6 cycloalkyl, halogen, wherein p and k are each independently selected from an integer of 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

[0045] In some embodiments, R is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropane, cyclobutane, cyclopentane, cyclohexane, fluorine, chlorine, bromine, iodine, wherein p and k are each independently selected from an integer of 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

[0046] In some embodiments, -L 5 -Selected from the following structures:

[0047]

[0048] The nitrogen end is connected to L 4 .

[0049] In some embodiments, the linker L is selected from:

[0050]

[0051]

[0052] Among them, the a end is covalently linked to the antibody part Ab, L 4 , R, and n are defined as above.

[0053] In some embodiments, the antibody-drug conjugate represented by formula (I) is selected from formula (I-1), formula (I-2) or (I-3):

[0054]

[0055] In some embodiments, the antibody-drug conjugate of formula (I) is selected from:

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] On the other hand, the present invention provides a linker-drug compound represented by formula (II), and its mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances:

[0064]

[0065] in,

[0066] L 1’ Selected from:

[0067]

[0068] L 2 , L 3 , L 4 , L 5 , R 1 , R 2 , R 3 The same as defined in formula (I).

[0069] In some embodiments, the linker-drug compound represented by formula (II) is selected from formula (II-1), formula (II-2) or formula (II-3):

[0070]

[0071] Where L 4 ,n,R,R 1 , R 2 The same as defined in formula (I).

[0072] In some embodiments, the linker-drug compound represented by formula (II) is selected from:

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] On the other hand, the present invention provides a compound represented by formula (III), and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance:

[0081]

[0082] Among them, R 1 , R 2 The same as defined in formula (I).

[0083] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of formula (I) of the present invention, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance thereof. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient and a carrier.

[0084] The present invention provides the antibody-drug conjugate of formula (I) of the present invention, and use of its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product in preparing a drug for treating or preventing tumors.

[0085] I. Definition of terms

[0086] Unless otherwise indicated, the following terms used in the present invention have the following meanings. When a trade name is used in the present invention, unless the context indicates otherwise, the trade name includes product formulations, generic drugs and active pharmaceutical ingredients of the trade name product.

[0087] The "linker-drug compound" of the present invention is connected to the antibody by conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reductive disulfide bond coupling and directional coupling (doi: 10.4161 / mabs.27436 ; doi: 10.1208 / s12248-014-9710-8). The present invention is preferably connected by reducing disulfide bond coupling between the light and heavy chains, that is, one or more of the disulfide bond sites between the light and heavy chains (two sites between the heavy chain, and two sites between the heavy chain and the light chain) are reduced to form a thiol group (sulfur atom of cysteine ​​residue) for reaction.

[0088] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically bind to tumor surface antigens, such as Farletuzumab, Trastuzumab, Pertuzumab, Nimotuzumab, Emibetuzumab, Besponsa, Pinatuzumab, Brentuximab, Gemtuzumab, Bevacizumab, cBR96 and Glematumamab or their antigen-binding fragments. The present invention preferably uses Farletuzumab to work.

[0089] The term "antibody-drug conjugate (ADC)" refers to a macromolecular biopharmaceutical in which a monoclonal antibody or antibody fragment is connected to a biologically active cytotoxin through a linker. At a certain concentration, the drug can not only target and kill tumor cells, but also exert a bystander effect and kill tumor cells with low or no expression.

[0090] The term "drug" refers to a cytotoxin, represented by D, which is a chemical molecule that has a strong ability to disrupt the normal growth of tumor cells.

[0091] The terms "linker" and "linker" refer to a chemical structure fragment that is connected to an antibody at one end and to a cytotoxin at the other end. It can also be connected to other linkers and then connected to a cytotoxin.

[0092] The term "cathepsin" refers to a cysteine ​​cathepsin present in the intracellular lysosomal environment. Cathepsin B has carboxypeptidase activity and can selectively cleave a series of dipeptides or polypeptides, such as Val-Cit, Val-Ala, and GGFG.

[0093] The term "peptide" refers to a short chain of amino acid monomers connected by peptide (amide) bonds.

[0094] The term "amino acid" refers to an organic compound that contains both an amino group and a carboxyl group in its molecular structure, and both the amino group and the carboxyl group are simultaneously connected to a -CH- structural unit. Amino acids can be divided into natural amino acids and unnatural amino acids. Natural amino acids include glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. Unnatural amino acids include citrulline, asparagine, etc.

[0095] The term "bystander effect" is when cytotoxins enter tumor cells to kill them and can cross the cell membrane to kill nearby tumor cells.

[0096] The term "stereoisomer" refers to compounds with the same chemical constitution but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography and / or fractional crystallization, based on the differences in the physicochemical properties of the components.

[0097] The term "mesomer" refers to a molecule containing asymmetric atoms, but with symmetry factors that make the molecule have an internal rotation of 0, i.e., no optical activity.

[0098] The term "racemate" refers to an equimolar mixture of an optically active chiral molecule and its enantiomer. It is composed of equal amounts of molecules with opposite optical rotation directions and equal optical rotation power. The optical activity of the molecules cancels each other out due to the interaction between these molecules, so it is optically inactive. In addition, although the physical properties of enantiomers are generally the same, the physical properties of the racemate, such as melting point and solubility, are often different from those of the corresponding enantiomers.

[0099] The term "enantiomers" refers to chiral molecules that are mirror images of each other. Enantiomers can be identified by the different directions in which they emit polarized light. The light can be right-handed or left-handed. Enantiomers are both optically active, one is left-handed and the other is right-handed, so enantiomers are also called optical isomers. Example: Lactic acid extracted from animal muscle is the right-handed isomer; lactic acid obtained by fermentation is the left-handed isomer. Synthetic lactic acid is the racemate.

[0100] The term “diastereomer” refers to a compound that has non-mirror image. Diastereomers are another form of stereoisomers, which have a ring structure. Diastereomers do not have any mirror image. Examples of diastereomers include double bond isomers, cis-trans isomers, and stereoisomers with several chiral centers.

[0101] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug compound or an antibody-drug conjugate). The compound may contain at least one amino or carboxyl group and may therefore form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, formates, benformates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, and the like. In addition, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter-acting examples. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter-acting atoms.

[0102] The term "solvate" means a physical association of the present compound with one or more solvent molecules, whether organic or inorganic. The physical association includes hydrogen bonding. In some cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to be separated. The solvent molecules in the solvate may exist in a regular arrangement and / or a disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0103] The term "isotopically labeled" means that the compound of the invention may be present in an isotopically traced or enriched form, containing one or more atoms whose atomic mass or mass number is different from the atomic mass or mass number of the atom found in the largest amount in nature. Isotopes may be radioactive or non-radioactive isotopes. Isotopes commonly used as isotope labels are: hydrogen isotopes, 2 H and 3 H; Carbon isotope: 13 C and 14 C; Chlorine isotope: 35 Cl and 37 Cl; Fluorine isotope: 18 F; Iodine isotope: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotope: 15 O, 17 O and 18 O and sulfur isotopes35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues.

[0104] The term "derivative" refers to a compound formed by replacing atoms or atomic groups in the molecule of a parent compound with other atoms or atomic groups, which is called a derivative of the parent compound.

[0105] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C1-C 10 alkyl), further preferably containing 1 to 8 carbon atoms (C1-C8 alkyl), and more preferably containing 1 to 6 carbon atoms (i.e. C1-C6 alkyl). 1-6 The term "alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl and the like.

[0106] The term "alkoxy" refers to -O-alkyl, the alkyl being as defined above, i.e., containing 1-20 carbon atoms, preferably, containing 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6) of an alkylthio group. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, and the like.

[0107] The term "alkylthio" refers to -S-alkyl, wherein the alkyl is as defined above, i.e., contains 1-20 carbon atoms, preferably, contains 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6) in the alkylthio group. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio, tert-butylthio, pentylthio, 1-methylbutylthio, 2-methylbutylthio, 3-methylbutylthio, 1,1-dimethylpropylthio, 1,2-dimethylpropylthio, 2,2-dimethylpropylthio, 1-ethylpropylthio, and the like.

[0108] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocyclic, bridged or spirocyclic ring. Preferably, it contains 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). The example of cycloalkyl includes but is not limited to cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, 2-ethyl-cyclopentane, dimethylcyclobutane, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0109] The term "aryl" refers to a monocyclic, bicyclic and tricyclic aromatic carbocyclic ring system containing 6-16 carbon atoms, or 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms, preferably 6-10 carbon atoms, and the term "aryl" can be used interchangeably with the term "aromatic ring". Examples of aryl groups may include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl or pyrenyl, etc.

[0110] The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing 5-14 members, or preferably 5-10 members, or preferably 5-8 members, and more preferably 5-6 members, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms are independently selected from O, N or S, and the number of heteroatoms is preferably 1, 2 or 3. Examples of heteroaryl include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothienyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

[0111] The term "halogen" refers to F, Cl, Br, I. The term "haloalkyl" refers to an alkyl group as defined above in which one, two or more hydrogen atoms or all hydrogen atoms are replaced by halogen. Representative examples of haloalkyl include CCl, CHCl, CHCl, CF, CHF, CHF, CBr, CHBr, CHBr, CI, CHI, CHI, CHCF, CFCF, etc.

[0112] The term "fluoromethyl" refers to monofluoromethyl, difluoromethyl, trifluoromethyl and the like groups.

[0113] The term "-(PEG) n -" means that the part has n -PEG-linked groups. For example, when the number of a linking group is 0, such as "-(CH)0-", it means that the linking group is a covalent bond.

[0114] The term "heterocyclic radical" refers to a non-aromatic ring that is fully saturated or partially unsaturated (non-fully unsaturated heteroaromatic) and can exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the heterocyclic ring is generally a 3 to 7 ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms, but excluding -OO-, -OS- or -SS-parts) independently selected from sulfur, oxygen and / or nitrogen. Non-limiting examples of heterocyclic radicals include but are not limited to oxirane, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, etc.

[0115] 2. Abbreviations

[0116] Table 1.

[0117]

[0118] DETAILED DESCRIPTION

[0119] The following is an explanation of the embodiments of the present invention by way of specific implementations. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0120] The test methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions, or parts are by weight.

[0121] Commercial reagents were purchased from Adamas, Aldrich, TCI, Titan, Energy Chemical and J&K Chemical and used as received. Unless otherwise stated, all reactions were carried out in dry glassware under a nitrogen atmosphere. Unless otherwise stated in the examples, the products were chromatographed by silica gel flash chromatography. Thin layer chromatography (TLC) used Silicycle 250mm silica gel F-254 plates. Unless otherwise stated, solutions refer to aqueous solutions. Unless otherwise stated, the reaction temperature was room temperature, 20°C to 30°C. The reaction progress in the examples was monitored by thin layer chromatography (TLC), the developing solvent used in the reaction, the system of eluents for column chromatography used to purify the compounds and the developing solvent system for thin layer chromatography, and the volume ratio of the solvents were adjusted according to the different polarities of the compounds; the stability of the eribulin derivatives can be adjusted by adding a small amount of weakly alkaline or neutral reagents such as aqueous ammonia, methanolic ammonia, and ammonium acetate to the developing solvent.

[0122] Example 1: Synthesis of B1-B12

[0123] Table 2.

[0124]

[0125]

[0126] Synthesis of intermediates 3a-3c: refer to the preparation described in patent WO2005077918A1, taking the synthesis of compound 3a as an example:

[0127]

[0128] 1a (745 mg, 5 mmol, 1.0 equiv., commercially available) and 2 (3.04 g, 10 mmol, 2.0 equiv.,) were added to a 50 mL reaction bottle equipped with a magnetic rod. After nitrogen was replaced three times, DMF (20 mL, commercially available) was added, stirred at room temperature for 3 minutes, and DIEA (1.77 mL, 10 mmol, 2.0 equiv., commercially available) was added dropwise to continue the reaction. After the reaction was complete, the reaction solution was concentrated and the residue was purified by silica gel chromatography to obtain a white solid 3a (1.33 g, yield 85%). MS (ESI): (M+H)+ calculated value 315.1, experimental value 315.2.

[0129] Table 3 Structures of compounds 3a-3c

[0130]

[0131] Intermediates 6a, 6b, 6e, and 6f were purchased commercially, and 6c-6d were prepared and synthesized according to https: / / doi.org / 10.1021 / ie401562j;

[0132] Table 4 Structures of compounds 6a-6f

[0133]

[0134] Take the 6c synthesis route as an example:

[0135]

[0136] At 25 °C, 8 (1.06 g, 10 mmol) and ZnO (0.13 mmol) were added to an open 250 mL single-necked bottle. After stirring for 15 min, the crude product was recrystallized from THF to give 6c, which was used directly in the next step without further purification.

[0137] Synthesis route of compound B1:

[0138]

[0139] Step 1: Add 4 (146.1 mg, 0.2 mmol, 1.0 equiv., commercially available) and 3a (94.3 mg, 0.3 mmol, 1.5 equiv.) to a 50 mL reaction bottle equipped with a magnetic rod, replace nitrogen three times, add THF (30 mL, commercially available), stir at room temperature for 3 minutes, add Et3N (83.2 uL, 0.6 mmol, 2.0 equiv., commercially available) dropwise to continue the reaction, after the reaction is complete, concentrate the reaction solution, and purify the residue by silica gel chromatography to obtain a white solid 5 (162.9 mg, yield 90%). MS (ESI): (M+H)+ calculated value 905.5, experimental value 905.6.

[0140] Step 2: Add trioxymethylene 6a (405.4 mg, 4.5 mmol, 90.0 equiv., commercially available) and phosphorus pentoxide (105.0 mg, 0.74 mmol, 14.8 equiv., commercially available) to a 50 mL reaction bottle equipped with a magnetic rod. After replacing nitrogen three times, add dichloromethane (8 mL, commercially available). Dissolve 5 (45.3 mg, 0.05 mmol, 1.0 equiv.) in 2 mL of dichloromethane and add to a 50 mL reaction bottle. After the reaction is completed, the reaction solution is filtered through a Buchner funnel, the filtrate is concentrated, dichloromethane (2 mL, commercially available) and petroleum ether (5 mL, commercially available) are added, and stirred at room temperature for 20 min. A yellow precipitate is generated in the system. The reaction solution is filtered through a funnel, and the reaction solution is concentrated to obtain a white solid B1 (33.5 mg, yield 73%). MS (ESI): (M+H)+ Calculated value 917.5, experimental value 917.6.

[0141] B2-B4, B6-B9 and B12 were prepared according to the method of B1, taking the synthesis of compound B7 as an example.

[0142]

[0143] Step 1: Add 4 (146.1 mg, 0.2 mmol, 1.0 equiv., commercially available) and 3b (98.4 mg, 0.3 mmol, 1.5 equiv.) to a 50 mL reaction bottle equipped with a magnetic rod, replace nitrogen three times, add THF (30 mL, commercially available), stir at room temperature for 3 minutes, add Et3N (83.2 uL, 0.6 mmol, 2.0 equiv., commercially available), continue the reaction, after the reaction is complete, concentrate the reaction solution, and purify the residue by silica gel chromatography to obtain a white solid 7 (161.7 mg, yield 88%). MS (ESI): (M+H)+ calculated value 919.5, experimental value 919.5.

[0144] Step 2: Add triacetaldehyde 6b (594 mg, 4.5 mmol, 90.0 equiv., commercially available) and phosphorus pentoxide (105.0 mg, 0.74 mmol, 14.8 equiv., commercially available) to a 50 mL reaction bottle equipped with a magnetic rod. After replacing nitrogen three times, add dichloromethane (8 mL, commercially available). Dissolve 7 (45.3 mg, 0.05 mmol, 1.0 equiv.) in 2 mL of dichloromethane and add to a 50 mL reaction bottle. After the reaction is completed, the reaction solution is filtered through a Buchner funnel, the filtrate is concentrated, dichloromethane (2 mL, commercially available) and petroleum ether (5 mL, commercially available) are added, and stirred at room temperature for 20 min. A yellow precipitate is generated in the system. The reaction solution is filtered through a funnel, and the reaction solution is concentrated to obtain a white solid B7 (39.2 mg, yield 83%). MS (ESI): (M+H) + Calculated value 945.5, experimental value 945.6.

[0145] The synthetic route of compound B5:

[0146]

[0147] To a 25 mL reaction bottle equipped with a magnetic bar, 5 (45.3 mg, 0.05 mmol, 1.0 equiv.), acetone (4 mL, commercially available) and 2,2-dimethoxypropane 6e (61.48 uL, 0.5 mmol, 10.0 equiv., commercially available) were added. After nitrogen bubbling for 15 minutes, PTSA (1.0 mg, 0.005 mmol, 0.1 equiv.) was added and the mixture was reacted at room temperature for 16 hours. The reaction solution was concentrated and the residue was purified by silica gel chromatography (PE / EA added with 10 nM NH3) to obtain a white solid B5 (39.2 mg, yield 83%). MS (ESI): (M+H) + Calculated value 945.5, experimental value 945.5.

[0148] B10-B11 refer to the preparation method of B5 above.

[0149] Synthesis route of compound B12:

[0150]

[0151] Step 1: Add 4 (146.1 mg, 0.2 mmol, 1.0 equiv., commercially available) and 3c (105.3 mg, 0.3 mmol, 1.5 equiv.) to a 50 mL reaction bottle equipped with a magnetic rod, replace nitrogen three times, add THF (30 mL, commercially available), stir at room temperature for 3 minutes, add Et3N (83.2 uL, 0.6 mmol, 2.0 equiv., commercially available) dropwise to continue the reaction, after the reaction is complete, concentrate the reaction solution, and purify the residue by silica gel chromatography to obtain a white solid 30 (141 mg, yield 75%). MS (ESI): (M+H)+ calculated value 943.5, experimental value 943.6.

[0152] Step 2: Add trioxymethylene 6a (405.4 mg, 4.5 mmol, 90.0 equiv., commercially available) and phosphorus pentoxide (105.0 mg, 0.74 mmol, 14.8 equiv., commercially available) to a 50 mL reaction bottle equipped with a magnetic rod. After replacing nitrogen three times, add dichloromethane (8 mL, commercially available). Dissolve 30 (45.3 mg, 0.05 mmol, 1.0 equiv.) in 2 mL of dichloromethane and add to a 50 mL reaction bottle. After the reaction is completed, the reaction solution is filtered through a Buchner funnel, the filtrate is concentrated, dichloromethane (2 mL, commercially available) and petroleum ether (5 mL, commercially available) are added, and stirred at room temperature for 20 min. A yellow precipitate is generated in the system. The reaction solution is filtered through a funnel, and the reaction solution is concentrated to obtain a white solid B12 (40.6 mg, yield 85%). MS (ESI): (M+H) + Calculated value 955.5, experimental value 955.5.

[0153] Example 2: Synthesis of C1-C5

[0154] Table 5.

[0155]

[0156]

[0157] The synthesis methods of C1-C4 are the same, taking the synthesis route of compound C1 as an example:

[0158]

[0159] Step 1: Add compounds 9 (188.2 mg, 1 mmol, 1.0 equiv., commercially available) and 10 (310.8 mg, 1.2 mmol, 1.2 equiv., commercially available) to a 10 mL reaction tube with a magnet. After replacing nitrogen three times, add a 1:1 volume ratio of 1,4-dioxane / water mixed solution (2 mL, commercially available) and Et3N (208.7 uL, 1.5 mmol, 1.5 equiv., commercially available) and stir at room temperature overnight. After the reaction is complete, add 1 M HCl aqueous solution to the reaction system, extract with ethyl acetate (20 mL×5, commercially available), combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate the filtrate under reduced pressure to obtain a white solid 11 (329.1 mg, yield 99%). MS (ESI): (M+H) + Calculated value 333.2, experimental value 333.2.

[0160] Step 2: Add 12 (275.5 mg, 0.99 mmol, 1.3 equiv., commercially available) and 11 (252.7 mg, 0.76 mmol, 1.0 equiv.) to a 25 mL reaction with a magnetic rod, replace nitrogen three times, add 3 mL of dichloromethane (commercially available), and place in an ice bath at 0°C. Dissolve DCC (204.0 mg, 0.99 mmol, 1.3 equiv. commercially available) and DAMP (9.3 mg, 0.076 mmol, 0.1 equiv., commercially available) in 2 mL of dichloromethane, then add to the reaction and continue the reaction overnight. After the reaction is completed, the reaction solution is filtered, the filtrate is concentrated, and the residue is purified by silica gel chromatography to obtain a white solid C1 (351.4 mg, yield 78%). MS (ESI): (M+H) + Calculated value 593.3, experimental value 593.4.

[0161] Synthesis of C5

[0162]

[0163] Step 1: Add 4-triisopropylsilyloxybenzaldehyde (50 g, 179.6 mmol, 1.0 equiv., commercially available) and THF (250 mL) to a 500 mL three-necked flask with a magnetic rod. Nitrogen was bubbled for 15 min at 0 ° C, and then NaBH4 (13.59 g, 359.1 mmol, 2.0 equiv., commercially available) was added in batches and the reaction was continued for 1 h. After the reaction was completed by TLC monitoring, 150 mL of ice water was added and stirring was continued for 30 min. Then, it was extracted with DCM (200 mL×3), and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain 13 (50 g, yield 100%). MS (ESI): (M+H)+ calculated value 281.2, experimental value 281.2.

[0164] Step 2: Add 13 (30 g, 107.0 mmol, 1.0 equiv.), di(p-nitrobenzene) carbonate 3a (48.8 g, 160.5 mmol, 1.5 equiv., commercially available) and DIEA (44.2 mL, 267.4 mmol, 2.5 equiv., commercially available) to a 500 mL three-necked flask with a magnetic rod and react at room temperature for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated and purified by column chromatography to obtain 14 (36.2 g, yield 76%). MS (ESI): (M+H) + Calculated value 446.2, experimental value 446.2.

[0165] Step 3: Add 14 (5 g, 11.2 mmol, 1.0 equiv., commercially available), 9 (2.3 g, 12.3 mmol, 1.1 equiv., commercially available) and THF and DMF (1:1; 30 mL) to a 50 mL reaction bottle equipped with a magnetic bar, and then add DIEA (4.35 g, 33.7 mmol, 5.9 mL, 3.0 equiv., commercially available) and react at 45 ° C for 5 h. After the reaction is complete as monitored by TLC, 15 is obtained without purification and directly used for the next step.

[0166] Step 4: Add 12 (4.0 g, 14.5 mmol, 1.3 equiv., commercially available) and 15 (5.5 g, 11.1 mmol, 1.0 equiv., commercially available) to a 25 mL reaction with a magnetic rod. After replacing nitrogen three times, add 150 mL of dichloromethane (commercially available) and place in an ice bath at 0°C. Dissolve DCC (3.0 mg, 14.5 mmol, 1.3 equiv. commercially available) and DAMP (135.4 mg, 0.111 mmol, 0.1 equiv., commercially available) in 15 mL of dichloromethane, then add to the reaction and continue the reaction overnight. After the reaction is completed, the reaction solution is filtered, the filtrate is concentrated, and the residue is purified by silica gel chromatography to obtain a white solid C5 (2.1 g, yield 25%). MS (ESI): (M+H) + Calculated value 755.4, experimental value 755.4.

[0167] Example 3: Synthesis of D1

[0168]

[0169] Step 1: Add methyl 3-(((tert-butoxycarbonyl)amino)methyl)benzoate 16 (3.2 g, 12.0 mmol, 1.0 equiv., commercially available) and methanol (50 mL, commercially available) into a 250 mL three-necked flask with a magnetic rod. After replacing the nitrogen three times, add hydrazine hydrate (1.7 mL, 2 mmol, 3.0 equiv., commercially available) and react at 75 ° C overnight. After the reaction is complete, add 100 mL of water to the reaction system, extract with ethyl acetate (100 mL×4, commercially available), and dry and concentrate the organic phase over anhydrous sodium sulfate to obtain a white solid 17 (3.1 g, yield 97%). MS (ESI): (M+H) + Calculated value 266.2, experimental value 266.3.

[0170] Step 2: Add 17 (3.0 g, 11.3 mmol, 1.0 equiv.) to a 100 mL two-necked bottle with a magnetic rod, and add KOH (762 mg, 13.6 mmol, 1.2 equiv., commercially available) and EtOH (30 mL, commercially available) in sequence. After nitrogen bubbling for 15 min, slowly add CS2 (0.99 mL, 1.5 equiv., commercially available). After the addition is complete, turn to 85 °C for reflux reaction. After the reaction is completed by TLC monitoring, the reaction solution is concentrated, and then 100 mL of water is added to dissolve and filter. The filtrate is adjusted to pH 4.0 with 3M HCl. Extract with ethyl acetate (100 mL×4, commercially available), and the organic phase is dried and concentrated over anhydrous sodium sulfate to obtain a white solid 18 (2.9 g, yield 82%). MS (ESI): (M+H) +Calculated value 308.1, experimental value 308.1.

[0171] Step 3: 18 (2.7 g, 8.8 mmol, 1.0 equiv.) and THF (2 mL, commercially available) were added to a 100 mL single-necked flask with a magnetic rod. Then, Et3N (2.7 g, 8.8 mmol, 1.0 equiv., commercially available) and iodomethane (1.4 g, 9.7 mmol, 1.1 equiv., commercially available) were added in turn under an ice bath. After 30 minutes, the reaction was moved to room temperature. After TLC monitoring, the reaction was complete, and then extracted with ethyl acetate (50 mL×5, commercially available). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a white solid 19 (1.8 g, yield 65%). MS (ESI): (M+H) + Calculated value 322.1, experimental value 322.2.

[0172] Step 4: 19 (1.7 g, 5.3 mmol, 1.0 equiv.) and ethyl acetate (20 mL, commercially available) were added to a 50 mL single-mouth bottle, and HCl (4 M HCl in ethyl acetate solution, 21.2 mmol, 4.0 equiv.) was added at room temperature. After reacting at room temperature for 5 hours, the reaction was completed as monitored by TLC, and the reaction solution was filtered to obtain a white solid 20 (1.4 g, yield 100%).

[0173] Step 5: 1,4-dioxane-2,6-dione 21 (1.9 g, 15.9 mmol, 1.1 equiv., commercially available), 20 (3.7 g, 14.5 mmol, 1.0 equiv.) and THF (50 mL, commercially available) were added sequentially into a 100 mL single-necked flask with a magnetic rod. After nitrogen bubbling for 15 min, Et3N (2.9 g, 4 mL, 2.0 equiv., commercially available) and H2O (1 mL, commercially available) were added sequentially. After reacting at room temperature for 1.5 h, TLC monitoring and LCMS showed that the reaction of the raw materials was basically complete. The reaction solution was concentrated to obtain a crude product 22. The crude product was washed with PE to remove some impurities, and the obtained solid was slurried with 50 mL of deionized water and filtered to obtain a clean product 22 (4.0 g, yield 82%). MS (ESI): (M+H) + Calculated value 338.1, experimental value 338.1.

[0174] Step 6: Add 22 (2.0 g, 5.9 mmol, 1.0 equiv.) and THF (50 mL, commercially available) to a 250 mL two-necked flask with a magnetic rod. After nitrogen bubbling for 15 min, add 30% H2O2 (5.4 g, 47.5 mmol, 8.0 equiv., commercially available) and ammonium heptamolybdate (1.0 g, 0.9 mmol, 0.15 equiv., commercially available) in turn. After 30 minutes, move to room temperature and react overnight. After the reaction is completed, add dimethyl sulfide (10.5 mL, 142.6 mmol, 24.0 equiv., commercially available) to the reaction solution, and continue the reaction for 3 h. A white precipitate appears in the reaction solution. Extract with ethyl acetate (100 mL×4, commercially available), and the organic phase is dried over anhydrous sodium sulfate and concentrated to obtain a solid. The solid is slurried with PE / EA to obtain a white solid D1 (1.4 g, yield 65%). MS (ESI): (M+H) + Calculated value 370.1, experimental value 370.1. Example 4: Compounds LD-1 to LD-24

[0175] Table 6

[0176]

[0177]

[0178] The synthesis methods of LD-1 to LD-24 are the same, taking the synthesis route of compound LD-1 as an example:

[0179]

[0180] Step 1: Add B1 (55.0 mg, 0.06 mmol, 1.0 equiv.) in THF / H2O (4:1, 8 mL, commercially available) to a 25 mL reaction bottle equipped with a magnetic rod, bubble for 15 min under a nitrogen atmosphere, then add C1 (142.3 mg, 0.24 mmol, 4.0 equiv.), and react at room temperature for 20 hours. After the reaction is complete, deionized water is added to the reaction system to quench, and it is extracted with ethyl acetate (containing 10 nM NH3, 3 mL×3, commercially available), the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The residue is separated by HPLC (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid 23 (37.6 mg, yield 52%). MS (ESI): (M+H) + Calculated value 1205.6, experimental value 1205.8.

[0181] Step 2: Add 23 (36.2 mg, 0.03 mmol, 1.0 equiv.) and tetrahydrofuran (4 mL, commercially available) to a 50 mL reaction bottle equipped with a magnetic rod. After nitrogen bubbling for 15 min, add TBAF (1 M in THF, 0.33 mL, 10 equiv.) and react at room temperature for 24 hours. After the reaction solution was concentrated, the residue was separated by HPLC (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid 24 (15.0 mg, yield 47%). (M+H) + Calculated value 1061.6, experimental value 1061.7.

[0182] Step 3: Add 24 (14.8 mg, 0.014 mmol, 1.0 equiv.) and 6-(maleimido)hexanoic acid succinimidyl ester (54.5 mg, 0.177 mmol, 12.6 equiv., commercially available) to a 50 mL reaction bottle equipped with a magnetic rod. After replacing nitrogen three times, add DMF (4 mL, commercially available) and triethylamine (5.8 uL, 0.042 mmol, 3.0 equiv., commercially available). After the reaction is complete, DMF is removed under vacuum and separated by HPLC preparation (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid LD-1 (6.1 mg, yield 35%). MS (ESI): (M+NH4) + Calculated value 1271.7, experimental value 1271.8.

[0183] Example 5: Compounds LD-25 to LD-29

[0184] Table 7

[0185]

[0186]

[0187] The synthesis methods of LD-25 to LD-29 are the same. Take the synthesis route of compound LD-25 as an example:

[0188]

[0189] 24 (9.6 mg, 0.009 mmol, 1.0 equiv.), D1 (6.6 mg, 0.018 mmol, 2.0 equiv.), DCC (3.7 mg, 0.018 mmol, 2.0 equiv.), HOBT (2.8 mg, 0.018 mmol, 2.0 equiv.) and DMF (2 mL, commercially available) were added to a 50 mL reaction bottle equipped with a magnetic bar. After nitrogen bubbling for 15 min, DIEA (3.0 uL, 0.018 mmol, 2.0 equiv commercially available) was added and reacted at room temperature overnight. After the reaction was completed, DMF was removed under vacuum and separated by HPLC preparation (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid LD-25 (8.0 mg, yield 63%). MS (ESI): (M+H) + Calculated value 1412.6, experimental value 1412.6.

[0190] Example 6: Compounds LD-30 to LD-32

[0191] Table 8

[0192]

[0193]

[0194] The synthesis methods of LD-30 and LD-31 are the same. Take the synthesis route of compound LD-30 as an example:

[0195]

[0196] Step 1: Add B12 (57.3 mg, 0.06 mmol, 1.0 equiv.) in THF / H2O (4:1, 6 mL) to a 25 mL reaction bottle equipped with a magnetic bar, bubble for 15 min under a nitrogen atmosphere, then add C5 (142.3 mg, 0.24 mmol, 4.0 equiv.), and react at 35 ° C for 16 hours. After the reaction is complete, add ammonia water (28%, 1 mL) to the reaction system, stir for 30 min, add deionized water to quench, extract with ethyl acetate (20 mL×3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue is purified by silica gel (mobile phase: A-DCM added with 10 nM NH3; B-MeOH) to obtain a white solid 25 (76.8 mg, yield 91%). MS (ESI): (M+H) + Calculated value 1405.8, experimental value 1405.9.

[0197] Step 2: Add 25 (42.2 mg, 0.03 mmol, 1.0 equiv.), cysteine ​​(18.2 mg, 0.15 mmol, 5.0 equiv.), and MeOH (2 mL) to a 50 mL reaction bottle equipped with a magnetic rod. Add K2CO3 (62.2 mg, 0.45 mmol, 15.0 equiv.) at 40 °C. After reacting for 2 h, filter out the solid in the reaction solution. After the filtrate is concentrated, the residue is separated by HPLC (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid 26 (20.8 mg, yield 63%). (M+H) + Calculated value 1099.6, experimental value 1099.7.

[0198] Step 3: Add 26 (15.4 mg, 0.014 mmol, 1.0 equiv.), D1 (7.7 mg, 0.021 mmol, 1.5 equiv.), DCC (5.8 mg, 0.028 mmol, 2.0 equiv.), HOBT (1.89 mg, 0.014 mmol, 1.0 equiv.), DIEA (2.3 uL, 0.014 mmol, 1.0 equiv.) and DMF (2 mL) to a 50 mL reaction bottle equipped with a magnetic rod. Stir overnight at 25 °C. After the reaction is completed, the residue is separated by HPLC (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) to obtain a white solid 27 (8.1 mg, yield 40%). MS (ESI): (M+H) + Calculated value 1450.6, experimental value 1450.7.

[0199] Step 4: 27 (8 mg, 0.0055 mmol, 1.0 equiv.), 28 (4.8 mg, 0.0083 mmol, 1.5 equiv.), sodium ascorbate (0.22 mg, 0.0011 mmol, 0.2 equiv.), THPTA (0.48 mg, 0.0011 mmol, 0.2 equiv.) and 27 (8 mg, 0.0055 mmol, 1.0 equiv.) were added to a 50 mL reaction bottle equipped with a magnetic rod. t BuOH / H2O (1:1; 2mL), nitrogen bubbling for 15min, then adding CuBr (0.16mg, 0.0011mmol, 0.2equiv., pretreated with dilute hydrochloric acid), reacting at room temperature for 1h. HPLC preparative separation (mobile phase: A-10nM NH4OAc; B-acetonitrile, gradient elution) gave a white solid LD-30 (5.8mg, yield 52%). MS (ESI): (M+H) + Calculated value 2037.0, experimental value 2037.2.

[0200] Example 7: Synthesis of LD-0

[0201]

[0202] 29 (117.5 mg, 0.15 mmol, 1.5 equiv.), 4 (73.0 mg, 0.1 mmol, 1.0 equiv.) and THF (5 mL) were added to a 50 mL reaction bottle equipped with a magnetic rod. After nitrogen bubbling for 15 min, Et3N (41.7 uL, 0.3 mmol, 3.0 equiv.) was added and reacted at room temperature for 12 h. HPLC preparative separation (mobile phase: A-10 nM NH4OAc; B-acetonitrile, gradient elution) gave a white solid LD-0 (103.1 mg, yield 75%). MS (ESI): (M+H) + Calculated value 1374.70, experimental value 1374.72.

[0203] Example 8: ADC sample preparation, general method for DAR determination, and cellular activity of ADC

[0204] The antibody sample was diluted to about 10 mg / mL with a suitable buffer (consistent with the sample buffer), and 2.2 to 2.5 equivalents of the reducing agent TCEP were added, with a target DAR of 4, and then the pH was adjusted to 7-7.4 with Tris buffer, and reduced at room temperature for 1-1.5 hours. The intermediate state of antibody reduction can be monitored by CE-SDS. After the antibody is fully reduced, an appropriate amount of saturated citric acid solution is first added to adjust the pH to about 6.5, and then an excess of linker-drug DMSO solution is added to make the linker-drug equivalent 6-10 times the antibody equivalent, and the coupling reaction is carried out at room temperature for about 30 minutes. After the coupling is completed, the reaction solution is first filtered, and then ultrafiltration is performed with a centrifugal concentrator to remove excess linker-drug and other small molecule impurities. After purification, the obtained sample is subjected to DAR determination by hydrophobic chromatography HIC or ultraviolet spectrophotometry. The data statistics of all ADC samples are shown in Table 9.

[0205] Table 9

[0206]

[0207]

[0208] The results showed that compared with MORAb-202, ADC-04, 05, 09, 14-18, 20, 24, and 28 had excellent cell killing effects on KB cells; other ADC samples had similar cell killing effects on KB cells.

[0209] The structure of MORAb-202 is as follows:

[0210]

[0211] Example 9: Cys-linker-Payload enzyme cleavage experiment

[0212] 1.CTB activation:

[0213] Cathepsin B lyophilized powder (10 units) was diluted to 10 UN / mL solution with the corresponding storage solution (pH = 5.0, 25mM acetate buffer + 1.0mM EDTA), the total volume was 1mL, and it was divided into 50uL / tube and stored at -80℃. When performing enzyme digestion reaction, take a 50uL CTB (10UN / mL) mother solution, add 860μL pH = 5.0 buffer, and then add 100uL Cys (200mM) activation solution, activate for 15min, the total volume is 1.0mL, and the final concentration of 0.5UN / mL CTB activation solution is obtained

[0214] 2. Blocking of substrate mother solution:

[0215] Measure 5 μL of 10mM LD-0, LD-1 or LD-30 DMSO solution, add 5 μL of 100mM (5.0equiv) Cys aqueous solution, mix well, and carry out blocking reaction for 5-10 minutes to obtain the blocked substrate mother solution Cys-LD-0, Cys-LD-1orCys-LD-30, whose substrate concentration is 10 μL 5mM.

[0216] 3. Perform enzyme digestion reaction:

[0217] The final concentration of the substrate is 40uM: Measure 2.0μL of 5mM substrate blocking solution, add 250μL of 0.5UN / mL CTB solution, the total volume is 0.25mL, incubate at 37℃, and sample and test at intervals of 30min until 24h.

[0218] 4. Test samples:

[0219] After the reaction time point, take an appropriate amount of sample, add an equal volume of ice-cold MeOH (HPLC grade) for dilution and precipitation, then centrifuge at more than 10000 rpm and 4°C for 10 min, and take the supernatant for detection.

[0220] 5. Data Results

[0221] The drug peak area detected without enzyme was taken as 1, and the percentage obtained by comparing the drug peak area detected by sampling at the corresponding time point with 1 was the drug release ratio at that point until 24 hours. Table 8 records the remaining amount of Cys-Linker-drug after 24 hours of Cys-LD-0, Cys-LD-1 or Cys-LD-30 digestion.

[0222] Table 10 Release of Cys-LD-0 / 1 / 30 under CTB enzyme conditions

[0223] Entry Cys-Linker-drug Remaining (24h) Cys-LD-0 5％ Cys-LD-1 7％ Cys-LD-30 3％

[0224] The experimental results showed that after 24 hours of enzyme digestion of Cys-LD-0, Cys-LD-1 or Cys-LD-30, the Cys-Linker-drug was almost completely cleaved and the released product was eribulin.

Claims

1. The antibody-drug conjugate represented by formula (I), and its mesomorph, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance: in, Ab is an antibody or an antigen-binding fragment thereof; m is an integer or decimal selected from 1 to 12; L is a linker moiety; R 1 , R 2 Each is independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxy, optionally substituted with the following substituents: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, C6-C 14 Aryl, 5-14 membered heteroaryl, or R 1 and R 2 Together with the carbon atom to which it is connected, it forms the following optionally substituted substituents: C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl, wherein the optionally substituted substituents are selected from one or more of halogen, halogenated C1-C3 alkyl, amino, cyano, nitro, hydroxyl, and hydroxy C1-C3 alkyl.

2. The antibody-drug conjugate of formula (I) according to claim 1, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, in, Ab is selected from antibodies or antigen-binding fragments thereof targeting FRα, HER2, HER3, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD73, CD105, EGFR, VEGFR, VEDF, PD-1, PD-L1, MET, RET, B7H3, B7H4, D113.

3. The antibody-drug conjugate of formula (I) according to claim 1 or 2, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, wherein the linker L is -L 1 -L 2 -L 3 -L 4 -L 5 -; in, -L 1 -Selected from: The a-end is covalently linked to the antibody part Ab, and the b-end is covalently linked to L 2 Covalently linked; R 3 selected from hydrogen, halogen, amino, cyano, nitro, hydroxyl, optionally substituted with the following substituents: C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10 Aryl, 5-10 membered heteroaryl; -L 2 - is selected from a bond or C1-C8 alkylene, C3-C8 cycloalkylene, 1-100-membered straight-chain heteroalkylene, 3-8-membered heterocyclylene, wherein the alkylene, cycloalkylene, heteroalkylene, heterocyclylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, NH2-C1-C6 alkyl, 1-6-membered heteroalkyl, C1-C6 alkoxy, halogen, hydroxyl, amino, nitro, cyano, carboxyl or C3-C8 cycloalkyl, and the heteroatoms in the heteroalkylene and heterocyclylene are selected from one or more of N, O or S; -L 3 - is selected from -C(O)-, -S(O)-, -S(O)O-; -L 4 - a peptide residue selected from 2 to 8 amino acids; -L 5 -Selected from: The nitrogen end is connected to L 4 , R is selected from hydrogen, C1-C8 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, halogen, and an optionally substituted hydrophilic side chain.

4. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 3, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled product, in, R 1 , R 2 Each is independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxyl, optionally substituted with the following substituents: C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylthio, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10 Aryl, 5-10 membered heteroaryl, or R 1 and R 2 Together with the carbon atom to which it is connected, it forms the following optionally substituted substituents: C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, wherein the optionally substituted substituents are selected from one or more of halogen, halogenated C1-C3 alkyl, amino, cyano, nitro, hydroxyl, and hydroxy C1-C3 alkyl.

5. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 4, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled product, in, R 1 , R 2 each independently selected from hydrogen, halogen, amino, cyano, nitro, hydroxy, an optionally substituted substituent selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, methylthio, ethylthio, n-propylthio, isopropylthio, cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, aziridine, azetidinyl, tetrahydropyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, phenyl, naphthyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, or R 1 and R 2 Together with the carbon atom to which it is attached, it forms the following optionally substituted substituents: cyclopropane, cyclobutane, cyclopentane, cyclohexane, aziridine, azetidinyl, tetrahydropyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, wherein the optionally substituted substituents are selected from one or more of fluorine, chlorine, bromine, iodine, monofluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, amino, cyano, nitro, hydroxyl, hydroxymethyl, hydroxyethyl.

6. The antibody-drug conjugate of formula (I) according to any one of claims 2 to 5, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled product, in, -L 2 - is selected from a bond, a C1-C8 alkylene, -(CH2CH2O)nCH2-, -(CH2CH2O)nCH2CH2-, -(CH2CH2O)nCH2CH2CH2-, wherein n is selected from an integer of 1-24 (e.g., an integer of 1-12, an integer of 3-8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24); -L 3 - is selected from -C(O)-; -L 4 - a peptide residue selected from 2 to 8 amino acids, wherein the amino acids are selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), cysteine ​​(Cys), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), citrulline (Cit), arginine (Arg) and histidine (His), wherein the nitrogen end is connected to L 3 ; -L 5 -Selected from: The nitrogen end is connected to L 4 , R is selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C3-C6 cycloalkyl, halogen, wherein p and k are each independently selected from an integer of 1-24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

7. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 6, and its mesoform, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled product, in, The linker L is selected from: Among them, the a end is covalently linked to the antibody part Ab, L 4 , R, and n are defined as above.

8. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 7, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, which is selected from:

9. A linker-drug compound represented by formula (II), and its mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: in, L 1’ Selected from: R 1 , R 2 Same as the definition in claim 1, L 2 , L 3 , L 4 , L 5 , R 3 The definition is the same as that of claim 3.

10. The linker-drug compound of formula (II) according to claim 9, and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, which is selected from formula (II-1), formula (II-2) or formula (II-3):

11. The linker-drug compound of formula (II) according to any one of claims 9 to 10, and its mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, which are selected from:

12. The compound represented by formula (III), and its racemate, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance: in, R 1 , R 2 The definition is the same as that of claim 1.

13. A pharmaceutical composition comprising the antibody-drug conjugate of formula (I) according to any one of claims 1 to 8, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance thereof.

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