Antibody-Eribulin or its derivative conjugates, intermediates, manufacturing methods, pharmaceutical compositions and uses thereof

Abstract

The present invention relates to an antibody-eribulin or a derivative thereof conjugate, its intermediate, preparation method, pharmaceutical composition and use. The antibody-eribulin or a derivative thereof conjugate represented by general formula I is provided as shown below. The conjugate of general formula I has high antitumor activity, high stability in circulation, and excellent tumor tissue targeting and therapeutic index. JPEG2025505809000211.jpg70127

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 2022101407965, filed on February 16, 2022. This application cites the above Chinese patent application in full.

[0002] The present disclosure relates to antibody-eribulin or derivative thereof conjugates, intermediates, methods of manufacture, pharmaceutical compositions and uses thereof. [Background technology]

[0003] Eribulin is a halichondrin-type microtubule dynamics inhibitor with unique binding properties. In addition to its mechanism of inhibiting microtubule dynamics growth, non-clinical studies have shown that eribulin exerts unique effects on the tumor microenvironment, such as increasing vascular perfusion and permeability in the tumor center, promoting epithelial status, and reducing the migration ability of breast cancer cells. Currently, eribulin has been approved for the treatment of breast cancer in more than 70 countries and regions, including Europe, America, and Asia. In the phase I clinical study reported by Synold et al., a total of 40 patients were enrolled, and 38 patients were evaluable. The administration method of eribulin mesylate was intravenous injection on days 1, 8, and 15, with a chemotherapy cycle of 28 days, and the maximum tolerated dose (MTD) was 1.4 mg / m 2 It was determined that the most common grade 3-4 side effect of eribulin mesylate treatment was neutropenia (64%). Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, which combine the tumor targeting effect of antibodies with the activity of biologically active molecules, making them biological missiles with very promising efficacy and safety advantages. The antibody guides the ADC to bind to the target cells, which are then taken up by the cells, and the small molecule drug is released into the cells through enzymatic degradation by the action of specific enzymes to treat the disease. Therefore, ADCs composed of eribulin and tumor-targeting antibodies are expected to eliminate or reduce the toxic side effects caused by eribulin acting on non-disease tissues while maintaining the therapeutic effect of eribulin, thereby improving the therapeutic effect.

[0004] Due to its high selectivity, high reactivity and excellent stability, maleimides are widely suitable for coupling with thiols of antibodies via Michael addition reaction to form ADC molecules. Of the 15 commercially available ADC drugs, 9 are conjugated via maleimides. Many literature reports have shown that thiosuccinimide bonds are unstable in the presence of thiol-containing substances (such as in plasma), and are either cleaved by reverse Michael reaction or exchanged with endogenous thiols such as albumin (HAS) and glutathione (GSH), resulting in suboptimal pharmacodynamics, pharmacokinetics and safety. Summary of the Invention

[0005] The object of the present disclosure is to provide an antibody-eribulin or derivative thereof conjugate, its intermediate, preparation method, pharmaceutical composition and use. The antibody-eribulin or derivative thereof conjugate of the present disclosure has high antitumor activity, can kill tumor cells more effectively, and has better in vivo antitumor effect than eribulin alone, ADC has high stability in circulation, can reduce the shedding of non-targeted bioactive molecules in non-targeted cells, and can increase the effective release of bioactive molecules in cells, the bioactive molecules have higher intratumoral and blood concentration ratios, and have a higher therapeutic index, and the addition of a basic nitrogen atom to the linker increases the overall exposure of the antibody-eribulin or derivative thereof conjugate in the relatively acidic tumor environment, improves tumor tissue targeting, increases the concentration ratio of bioactive molecules in the tumor and blood, and has a higher therapeutic index.

[0006] To this end, in a first aspect of the present disclosure, the present disclosure provides an antibody-eribulin or a derivative thereof conjugate represented by formula I, or a stereoisomer of said conjugate, a prodrug thereof, a pharma-ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof.

[0007] [ka]

[0008] however, Tb is an antibody or antigen-binding fragment thereof bound to a target, or a targeting moiety; q is the drug-Tb coupling ratio, R 1 , R 2 are each independently selected from hydrogen (H), deuterium (D), C1-6 alkyl, or R 1 and R 2 form a 5- to 6-membered heterocycle together with the nitrogen atom and oxygen atom linked thereto, and the 5- to 6-membered heterocycle is optionally substituted by one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, and C3-6 heterocyclyl;

[0009] L1 is [ka] wherein position 1 is linked to Tb via an S atom and position 2 is linked to L2;

[0010] R c is selected from hydrogen (H), C1-6 alkyl,

[0011] L2 is [ka] wherein position 1 is linked to L1 and position 2 is linked to L3;

[0012] p is selected from any integer between 0 and 10; Y is selected from -CH2-, -OCH2CH2-; Z is -CR 3 R 4 -, -NR 5 - selected from R 3 , R 4are each independently selected from hydrogen (H), C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, and C3-6 heterocyclyl; or R 3 and R 4 form a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring together with the carbon atom to which they are linked, R 5 is selected from hydrogen (H), C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C3-6 heterocyclyl;

[0013] L3 is a chemical bond, [ka] , an amino acid residue, or a short peptide consisting of 2 to 10 amino acid residues, wherein the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or an AA 1 or a stereoisomer thereof,

[0014] AA 1 The structure of the amino acid residue represented by is as shown below.

[0015] [ka]

[0016] however, R a , R b are each independently hydrogen (H) and [ka] and R a , R b At the same time, it is not hydrogen (H), Or, R a and R btogether with the carbon atom to which they are attached form a 4-10 membered heterocyclic ring, said 4-10 membered heterocyclic ring optionally containing one or more R 0 is replaced by

[0017] r,r 1 are each independently selected from any integer between 0 and 20;

[0018] R m1 , R n1 are each independently selected from hydrogen (H), C1-6 alkyl, and C3-6 cycloalkyl; Or, R m1 and R n1 form a 4- to 10-membered heterocyclic ring together with the nitrogen atom to which they are linked, and the 4- to 10-membered heterocyclic ring may optionally be one or more R 0’ is replaced by

[0019] R 0 , R 0’ are each independently C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 and 4-10 membered heterocyclyl optionally substituted with C1-6 alkyl;

[0020] R m2 , R n2 are each independently selected from hydrogen (H) and C1-6 alkyl;

[0021] L4 is absent or present, and if present, L4 is [ka] and wherein position 1 is linked to L3 and position 2 is linked to the nitrogen atom of eribulin.

[0022] In some embodiments, the antibody-eribulin or derivative thereof conjugate of formula I, or a stereoisomer of the conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

[0023] however, Tb is a target-bound antibody or antigen-binding fragment thereof, or a targeting moiety; q is the coupling ratio of the drug Tb, R 1 , R 2 are each independently selected from hydrogen (H), deuterium (D), C1-6 alkyl, or R 1 and R 2 form a 5- to 6-membered heterocycle together with the nitrogen atom and oxygen atom connected thereto, and the 5- to 6-membered heterocycle is optionally substituted by one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, and C3-6 heterocyclyl;

[0024] L1 is [ka] wherein position 1 is linked to Tb via an S atom and position 2 is linked to L2;

[0025] R c is selected from hydrogen (H), C1-6 alkyl,

[0026] L2 is

[0027] [ka] wherein position 1 is linked to L1 and position 2 is linked to L3;

[0028] p is selected from any integer between 0 and 10; Y is selected from CH2, OCH2CH2; Z is CR 3 R 4 , N.R. 5 is selected from

[0029] R 3 , R 4 are each independently selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, and C3-6 heterocyclyl; or R 3 and R 4 form a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring together with the carbon atom to which they are linked,

[0030] R 5 is selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C3-6 heterocyclyl;

[0031] L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues, and the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or AA 1 or a stereoisomer thereof,

[0032] AA 1 The structure of the amino acid residue represented by is as shown below.

[0033] [ka]

[0034] however, R a , R b are each independently H and [ka] and R a , R b is not H at the same time, Or, Ra and R b together with the carbon atom to which they are attached form a 4-10 membered heterocyclic ring, said 4-10 membered heterocyclic ring optionally containing one or more R 0 is replaced by

[0035] r,r 1 are each independently selected from any integer between 0 and 20;

[0036] R m1 , R n1 are each independently selected from H, C1-6 alkyl, and C3-6 cycloalkyl; Or, R m1 and R n1 form a 4- to 10-membered heterocyclic ring together with the nitrogen atom to which they are linked, and the 4- to 10-membered heterocyclic ring may optionally be one or more R 0’ is replaced by

[0037] R 0 , R 0’ are each independently C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 and 4-10 membered heterocyclyl optionally substituted with C1-6 alkyl;

[0038] R m2 , R n2 are each independently selected from H and C1-6 alkyl;

[0039] L4 is absent or present, and if present, L4 is [ka] and wherein position 1 is linked to L3 and position 2 is linked to the nitrogen atom of eribulin.

[0040] In some implementations, L1 [ka] and position 1 is linked to Tb via an S atom and position 2 is linked to L2, L3 is not a chemical bond, Val-Cit, Val-Lys, (Gly)2-Phe-Gly or Ala-Ala-Asn, or L4 exists but [ka] isn't it.

[0041] In some implementations, L1 [ka] wherein position 1 is linked to Tb via an S atom, position 2 is linked to L2, and when L3 is Val-Cit, Val-Lys, (Gly)2-Phe-Gly or Ala-Ala-Asn, L4 is absent; [ka] Alternatively, If L3 is a chemical bond, then L4 is present.

[0042] In some embodiments, q is selected from any number between 0.1 and 16.0, and in preferred embodiments, q is selected from any integer between 0.1 and 10.0.

[0043] In some embodiments, q is selected from any number between 2 and 8.

[0044] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0045] In some implementations, R 1 , R2 is hydrogen (H) or R 1 and R 2 together with the nitrogen and oxygen atoms connected to them. [ka] where position 1 is linked to L4 or L3.

[0046] In some implementations, L1 is [ka] wherein position 1 is linked to Tb via an S atom and position 2 is linked to L2.

[0047] In some implementations, L1 is [ka] wherein position 1 is linked to Tb via an S atom and position 2 is linked to L2.

[0048] In some implementations, L1 is [ka] wherein position 1 is linked to Tb via an S atom and position 2 is linked to L2.

[0049] In some implementations, L1 is [ka] where position 1 is linked to Tb via an S atom and position 2 is linked to L2.

[0050] In some implementations, R c is hydrogen (H).

[0051] In some embodiments, p is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8.

[0052] In some embodiments, Z is -CH2-, -C(CH3)2-, or -NH-.

[0053] In some embodiments, Z is -C(CH3)2-.

[0054] In some implementations, R 3 , R 4 are each independently selected from hydrogen (H) and methyl.

[0055] In some implementations, R 5 is hydrogen (H) or methyl.

[0056] In some implementations, L2: [ka] wherein position 1 is linked to L1 and position 2 is linked to L3.

[0057] In some implementations, L2: [ka] wherein position 1 is linked to L1 and position 2 is linked to L3.

[0058] In some implementations, L2: [ka] where position 1 is linked to L1 and position 2 is linked to L3.

[0059] In some embodiments, L3 is selected from the group consisting of the amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, and AA. 1 or 2 to 10 Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AA 1 The peptide is selected from short peptides consisting of amino acid residues selected from:

[0060] In some embodiments, L3 is at least one (e.g., one, two, or three) AA 1 or a stereoisomer thereof.

[0061] In some embodiments, L3 is at least one AA 1 or a stereoisomer thereof.

[0062] In some implementations, L3 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AA 1 , Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA 1 , Ala-AA 1 , Gly-AA 1 , A.A. 1 -Gly, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Asp, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1-Gly, Lys-(Ala)2-Asn, Lys-(Ala)2-Asp, Gly-Phe-Gly, (Gly)2-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Ar g, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-(Gly)2, Val-Lys, Lys-Ala-Asn, (Gly)2-Phe-AA 1 is selected from.

[0063] In some implementations, L3 is Val-AA 1 -Gly, Val-AA 1 , Val-Cit, (Gly)2-Phe-AA 1 is selected from.

[0064] In some implementations, L3 is Val-AA 1 and Val-Cit.

[0065] In some embodiments, L3 is a chemical bond, [ka] and wherein position 1 is linked to L2 and position 2 is linked to L4 or a nitrogen atom of eribulin.

[0066] In some implementations, L3 is [ka] and wherein position 1 is linked to L2 and position 2 is linked to L4 or a nitrogen atom of eribulin.

[0067] In some implementations, L3 is [ka] and wherein position 1 is linked to L2 and position 2 is linked to L4 or a nitrogen atom of eribulin.

[0068] In some implementations, L3 is [ka] and wherein position 1 is linked to L2 and position 2 is linked to L4 or a nitrogen atom of eribulin.

[0069] In some implementations, R a , R b Among them, any one is hydrogen (H) and the other one is [ka] It is.

[0070] In some implementations, R a and R b R together with the carbon atoms connected to them 0 Forms a 5- to 6-membered heterocycle substituted by

[0071] In some implementations, R a and R b R together with the carbon atoms connected to them 0 The compound forms a piperidine or piperazine ring substituted by:

[0072] In some implementations, R a and R b R together with the carbon atoms connected to them 0 The compound forms a piperidine ring substituted by

[0073] In some implementations, R a and R b together with the carbon atoms connected to them [ka] The first carbon atom is R a and R b is a carbon atom connected together with

[0074] In some implementations, r, r 1 are each independently selected from 0, 1, 2, 3, 4, and 5.

[0075] In some implementations, r, r 1 are each independently selected from 0 and 4.

[0076] In some embodiments, r is 0 and r 1 is 4.

[0077] In some implementations, R m1 , R n1 are each independently selected from hydrogen (H) and C1 to C6 alkyl, preferably C1 to C6 alkyl.

[0078] In some implementations, R m1 , R n1 are each independently selected from hydrogen (H), methyl, ethyl, n-propyl and n-butyl, preferably methyl, ethyl, n-propyl and n-butyl.

[0079] In some implementations, R m1 and R n1 are optionally R together with the nitrogen atom connected thereto. 0’ Forms a 5- to 6-membered heterocycle substituted by

[0080] In some implementations, R m1 and R n1 are optionally R together with the nitrogen atom connected thereto. 0’ The compound forms a piperidine or piperazine ring substituted by:

[0081] In some implementations, R m1 and R n1 together with the nitrogen atoms connected to them [ka] The first nitrogen atom is R m1 and R n1 is a nitrogen atom linked together with

[0082] In some implementations, R 0 , R 0’ are each independently C1-6 alkyl, -NR m2 R n2 and 5-6 membered heterocyclyl optionally substituted by C1-6 alkyl.

[0083] In some implementations, R 0 is selected from C1-6 alkyl and 5-6 membered heterocyclyl substituted by C1-6 alkyl, said 5-6 membered heterocyclyl being selected from piperidinyl and piperazinyl.

[0084] In some implementations, R 0 is selected from methyl, ethyl, and 5- to 6-membered heterocyclyl substituted by methyl, said 5- to 6-membered heterocyclyl being piperidinyl.

[0085] In some implementations, R 0 is selected from methyl and 5- to 6-membered heterocyclyl substituted by methyl, said 5- to 6-membered heterocyclyl being piperidinyl.

[0086] In some implementations, R 0 is methyl, ethyl and [ka] is selected from.

[0087] In some implementations, R 0 is methyl and [ka] is selected from.

[0088] In some implementations, R 0’ is C1-6 alkyl and -NR m2 R n2 is selected from.

[0089] In some implementations, R 0’ -Methyl and -NR m2 R n2 is selected from.

[0090] In some implementations, R m2 , R n2 is methyl.

[0091] In some implementations, AA 1 The amino acid residue represented by [ka] For example, [ka] It is.

[0092] In some implementations, AA 1 The amino acid residue represented by [ka] For example, [ka] It is.

[0093] In some implementations, AA 1 The amino acid residue represented by [ka] It is.

[0094] In some embodiments, L4 is absent or present, and when L4 is present, L4 is [ka] and wherein position 1 is linked to L3 and position 2 is linked to the nitrogen atom of eribulin.

[0095] In some implementations, L4 is [ka] where position 1 is linked to L3 and position 2 is linked to the nitrogen atom of eribulin.

[0096] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0097] [Table 1]

[0098] However, Y, Z, p, and AA 1is as defined in any one of the embodiments of the present disclosure, and position 1 is linked to Tb via an S atom and position 2 is linked to the nitrogen atom of eribulin.

[0099] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0100] [Table 2]

[0101] However, Y, Z, p, and AA 1 is as defined in any one of the embodiments of the present disclosure, and position 1 is linked to Tb via an S atom and position 2 is linked to the nitrogen atom of eribulin.

[0102] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0103] [Table 3]

[0104] However, Y, Z, p, and AA 1 is as defined in any one of the embodiments of the present disclosure, and position 1 is linked to Tb via an S atom and position 2 is linked to the nitrogen atom of eribulin.

[0105] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0106] [Table 4-1] [Table 4-2] [Table 4-3]

[0107] Position 1 is linked to Tb via a S atom and position 2 is linked to the nitrogen atom of eribulin.

[0108] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0109] [Table 5-1] [Table 5-2]

[0110] Position 1 is linked to Tb via a S atom and position 2 is linked to the nitrogen atom of eribulin.

[0111] In some embodiments, the linker [ka] is selected from the formulas represented below:

[0112] [Table 6]

[0113] Position 1 is linked to Tb via a S atom and position 2 is linked to the nitrogen atom of eribulin.

[0114] In some embodiments, the antibody-eribulin or derivative thereof conjugate is preferably a compound described below.

[0115] [ka]

[0116] [ka]

[0117] [ka]

[0118] [ka]

[0119] [ka]

[0120] [ka]

[0121] Furthermore, one of ordinary skill in the art should understand that L is linked to a sulfhydryl contained in Tb (e.g., an antibody) itself after disulfide bond opening (e.g., reduction of the disulfide bond with the reducing agent TCEP opens the disulfide bond to generate sulfhydryl-SH), i.e., -S- between L and Tb is not an additional external sulfur atom. For example, -S- is not an additional external sulfur atom, but is -S- formed when a sulfhydryl contained in Tb itself is bound to L after disulfide bond opening.

[0122] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a probody, a bispecific antibody, or a multispecific antibody.

[0124] In some embodiments, Tb is a monoclonal antibody or an antigen-binding fragment thereof.

[0125] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that has endocytotic activity.

[0126] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that has activity of binding to free antigens in tumor tissue and / or tumor cell surface antigens.

[0127] In some preferred embodiments, Tb is selected from an antibody or antigen-binding fragment thereof that binds to the following targets: epidermal growth factor, integrin alpha5beta6, integrin alpha4beta7, 0772P, 5T4, ACTA2, ADGRE1, AGS-16, AIF1, AKR1C1, AKR1C2, ANGPTL4, ApoE, ASLG659, BAFF-R, BMPR1B, BNIP3, Brevican, C1QA, C1QB, CA6, pCAD, P-cadherin, CADM1, CCL5, CCR5, CDl lb, CD1 lc, CD19, CD20, CD21, CD22, CD30, CD33, CD37, CD40, CD45 (PTPRC), CD49D. (ITGA4), CD56, CD66e, CD70, CD72, CD74, CD79a, CD80, CD138, CD223, CDCP1, CDH11, Claudin18.2, COL6A3, COL7A1, CRIPTO, CSF1R, CTGF, CTSD, CTSS, CXCL10, CXCL11, CXCR5, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, B7H3, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, Endothelin receoptor, ENPP3, EpCAM, EphB2R, ETBR, FGF2, FGFR2, FcRH1, FcRH2, GEDA, GPNMB, GZMB, Her2, Her3, HLA-DOB, HMOX1, IFNG, IFI6, IGF-1R, IGFBP3, IL10RA1, IL20 Rα, IL-6, IRTA2, KISS1R, KRT33A, LOX, LRRC15, LUM, LY6E, LY64, LY86, LYPD3, MCPT8, MDP, Mesothelin, c-Met, MFI2, MMP10, MMP14, MMP16, MPF, MSG783, MS4A7, MUC 1, MUC16, NaPi2b, NaPi3b, NCA, Nectin 4, NOG, P2X5, PD-L1, PDK1, PDGFRA, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCA hlg, PSMA, RNF43, ROR1, Sema5b, SERPINEl, SLC39A6, SLITRK6, SLTRK6, STAT1, STC2, STEAP1, STEAP2, TCF4, TENB2, TGF, TGFBR1, TGFB2, tissue factor (Tissue factor), TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA and WNT5A. .

[0128] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-Trop-2 antibody or antigen-binding fragment thereof, an anti-Her2 antibody or antigen-binding fragment thereof, an anti-Her3 antibody or antigen-binding fragment thereof, or an anti-EGFR antibody or antigen-binding fragment thereof.

[0129] In some embodiments, Tb is an anti-Her2 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-Her2 antibody is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0130] In some embodiments, Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-Trop-2 antibody is datopotamab, sacituzumab, or an antigen-binding fragment thereof.

[0131] In some embodiments, Tb is an anti-EGFR antibody or an antigen-binding fragment thereof. In some embodiments, the anti-EGFR antibody is demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzuotuximab, zalutumumab, cetuximab, or an antigen-binding fragment thereof.

[0132] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-B7H3 antibody is enoblituzumab, mirzotamab, omburtamab, 1D1-01, 2E3-02 antibody or an antigen-binding fragment thereof.

[0133] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof.

[0134] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs), where the CDRs are defined by the Kabat numbering system: HCDR1, whose sequence is SEQ ID NO:9; HCDR2, whose sequence is SEQ ID NO:10; HCDR3, whose sequence is SEQ ID NO:11; and / or LCDR1, whose sequence is SEQ ID NO:12; LCDR2, whose sequence is SEQ ID NO:13; LCDR3, whose sequence is SEQ ID NO:14.

[0135] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs), where the CDRs are defined by the IMGT numbering system:

[0136] an HCDR1 whose sequence is SEQ ID NO:15, an HCDR2 whose sequence is SEQ ID NO:16, an HCDR3 whose sequence is SEQ ID NO:17; and / or an LCDR1 whose sequence is SEQ ID NO:18, an LCDR2 whose sequence is GAS, and an LCDR3 whose sequence is SEQ ID NO:19.

[0137] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO: 1 or 5, and / or a VL set forth in SEQ ID NO: 2 or 6. It further comprises (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof; and / or (b) a human immunoglobulin light chain constant region (CL) or a variant thereof.

[0138] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:5, a CH set forth in SEQ ID NO:20 or a variant thereof, and / or a VL set forth in SEQ ID NO:6, a CL set forth in SEQ ID NO:21 or a variant thereof.

[0139] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO: 1, a CH set forth in SEQ ID NO: 20 or a variant thereof, and / or a VL set forth in SEQ ID NO: 2, a CL set forth in SEQ ID NO: 21 or a variant thereof.

[0140] In some preferred embodiments, the B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain set forth in SEQ ID NO:7 and / or a light chain set forth in SEQ ID NO:8.

[0141] In some preferred embodiments, the B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain set forth in SEQ ID NO:3 and / or a light chain set forth in SEQ ID NO:4.

[0142] In some embodiments, the antibody-eribulin or derivative thereof conjugate is preferably a compound described below.

[0143] [ka]

[0144] Here, B7H3-mAb is the 2E3-02 antibody, and q is selected from any integer between 0.1 and 10.0, and preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0145] In some embodiments, the antibody-eribulin or derivative thereof conjugate is preferably a compound described below.

[0146] [ka]

[0147] Here, q is selected from any integer between 0.1 and 10.0, and preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0148] In some embodiments, the antibody-eribulin or derivative thereof conjugate is preferably a compound described below.

[0149] [ka]

[0150] Here, B7H3-mAb is the 2E3-02 antibody, and q is selected from any integer between 0.1 and 10.0, and preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0151] In some embodiments, the antibody-eribulin or derivative thereof conjugate is preferably a compound described below.

[0152] [ka] .

[0153] However, the antibody is Trastuzumab, and q is selected from any integer between 0.1 and 10.0, and preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0154] The present disclosure further provides a drug linker conjugate represented by general formula II, or a stereoisomer of said drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

[0155] [ka]

[0156] however, L1 [ka] then position 1 is linked to Lg and position 2 is linked to L2; Lg is a leaving group when reacting with Tb, and L2, L3, L4, R c , R 1 and R 2 is as defined in any one of the embodiments of the present disclosure;

[0157] L1 [ka] If so, Lg-L1 is [ka] and L2, L3, L4, R c , R 1 and R 2 is as defined in any one of the embodiments of the present disclosure.

[0158] In some embodiments, Lg is a halogen, a sulfone, a tertiary amine (MeN + , Et3N + ), diazonium, -OMs, MeSO2-, CF3SO3-.

[0159] In some embodiments, Lg is selected from F, Cl, and MeSO2-.

[0160] In some embodiments, Lg is selected from F, MeSO2-.

[0161] In some embodiments, the drug linker conjugate represented by general formula II is preferably any one of the following compounds:

[0162] [ka]

[0163] [ka]

[0164] [ka]

[0165] [ka]

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] In some embodiments, the drug linker conjugate is preferably the compound below:

[0171] [ka]

[0172] The present disclosure further provides a linker having the general formula III:

[0173] [ka]

[0174] Lg, L1, L2, L3 and L4 are as defined in any one of the embodiments of the present disclosure, and Lg1 is a leaving group when reacting with eribulin or a derivative thereof.

[0175] In some embodiments, Lg1 is preferably -OH, [ka] Or a halogen.

[0176] The present disclosure provides a method for preparing the antibody-eribulin or derivative thereof conjugate, its stereoisomer, its pharma- ceutically acceptable salt, or its pharma- ceutically acceptable solvate, the method comprising coupling a sulfhydryl on Tb with a drug-linker conjugate represented by the general formula II: [ka] where Tb, q, Lg, L1, L2, L3, L4, R1 and R2 are as defined in any one of the embodiments of the present disclosure;

[0177] In some embodiments, the method comprises a step of coupling a sulfhydryl on Tb with a drug-linker conjugate represented by formula II under suitable solvent and conditions to form a CS bond.

[0178] [ka]

[0179] In some embodiments, the molar ratio of sulfhydryls on the Tb to the drug-linker conjugate can be 1:(1-20), e.g., 1:(2-20), 1:(4-20), 1:(6-20), 1:(8-20), 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20), or 1:(18-20).

[0180] In some embodiments, the coupling reaction is preferably carried out in water and / or an organic solvent, preferably one or more of N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, nitrile-based solvents (e.g., acetonitrile) and alcohol-based solvents (e.g., methanol, ethanol).

[0181] In some embodiments, the work-up procedure of the method may be a conventional work-up procedure for coupling reactions in the art, preferably purifying the coupling product by chromatography, which is usually one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography.

[0182] The present disclosure provides a method for preparing a drug-linker conjugate represented by formula II, selected from any one of the following methods:

[0183] Method 1 includes carrying out the reaction shown below between a linker represented by general formula III and eribulin or a derivative thereof, [ka] Lg, L1, L2, L3, L4, and Lg1 are as defined in any one of the embodiments of the present disclosure, wherein eribulin or a derivative thereof comprises a group reactive with Lg1;

[0184] Method 2 includes carrying out a coupling reaction between a compound represented by general formula III′ and a compound represented by general formula III″ as shown below: [ka] Lg, L1, L2, L3, L4, R1 and R2 are as defined in any one of the embodiments of the present disclosure, and L3' and L4' are reacted to obtain L3-L4.

[0185] In one preferred embodiment, the method for preparing a drug-linker conjugate represented by formula II of the present disclosure includes activating an A fragment, followed by carrying out a coupling reaction with a C fragment as shown below, to obtain a coupling product D, which is a drug-linker conjugate represented by formula II.

[0186] [ka]

[0187] Antibody-Eribulin or its derivative conjugates The present disclosure provides a group of antibody-eribulin or derivative thereof conjugates, including the antibody-eribulin or derivative thereof conjugate, a stereoisomer thereof, a pharma- ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, wherein the antibody-eribulin or derivative thereof conjugate in the mixture has two or more q values.

[0188] In certain embodiments, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from integers or decimals of 1 to 10;

[0189] In certain embodiments, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from integers or decimals of 1 to 8;

[0190] In certain embodiments, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from integers or decimals from 2 to 8;

[0191] In certain embodiments, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from the following integers or decimals: 2±0.5, 4±0.5, 6±0.5, and 8±0.5;

[0192] In certain preferred embodiments, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.2, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.

[0193] In one embodiment, the ADC antibody-eribulin or derivative thereof conjugates include ADCs with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 payload types). It should be noted that degradation products may be generated, such that the antibody-eribulin or derivative thereof conjugates may also include DARs of 1, 3, 5, and 7. Furthermore, the ADCs of the antibody-eribulin or derivative thereof conjugates may have a DAR greater than 8. The ADC antibody-eribulin or derivative thereof conjugates are generated by interchain disulfide reduction and coupling.

[0194] In one embodiment, the ADC antibody-eribulin or derivative thereof conjugates include both ADCs with a DAR of 4 or lower (i.e., 4 or fewer payload types) and ADCs with a DAR of 6 or higher (i.e., 6 or more payload types).

[0195] The present disclosure provides a pharmaceutical composition comprising the above-mentioned antibody-eribulin or derivative thereof conjugate, its stereoisomer, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the above-mentioned drug linker conjugate represented by formula II, its stereoisomer, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the above-mentioned antibody-eribulin or derivative thereof conjugates, and one or more optional pharmaceutical auxiliary agents. The above-mentioned antibody-eribulin or derivative thereof conjugate, its stereoisomer, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the above-mentioned drug linker conjugate represented by formula II, its stereoisomer, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the above-mentioned antibody-eribulin or derivative thereof conjugates may be in a therapeutically effective amount.

[0196] The present disclosure provides a pharmaceutical composition, in which when the antibody-eribulin or a derivative thereof-containing conjugate has only one q value, the ratio (DAR) of the conjugate of eribulin or a derivative thereof to the antibody corresponds to the q value. In some embodiments, the ratio (DAR) of the conjugate of eribulin or a derivative thereof to the antibody is selected from 1 to 8. In some preferred embodiments, the ratio (DAR) of the conjugate of eribulin or a derivative thereof to the antibody is selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0197] The present disclosure provides the use of the above-mentioned antibody-eribulin or derivative thereof conjugate, its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate, or the above-mentioned drug linker conjugate represented by formula II, its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate, or the above-mentioned antibody-eribulin or derivative thereof conjugates, or the above-mentioned pharmaceutical composition, in the manufacture of a medicament for treating and / or preventing a disease associated with abnormal cell activity (e.g., cancer disease). The above-mentioned antibody-eribulin or derivative thereof conjugate, its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate, or the above-mentioned drug linker conjugate represented by formula II, its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate, or the above-mentioned antibody-eribulin or derivative thereof conjugates, or the above-mentioned pharmaceutical composition may be in a therapeutically effective amount.

[0198] The present disclosure provides a method for preventing and / or treating a disease associated with abnormal cellular activity (e.g., a cancer disease), comprising administering to an individual in need thereof a prophylactically and / or therapeutically effective amount of the above-mentioned antibody-eribulin or derivative thereof conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the above-mentioned drug linker conjugate represented by formula II, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the above-mentioned antibody-eribulin or derivative thereof conjugates, or the above-mentioned pharmaceutical composition.

[0199] In some embodiments, the disease associated with abnormal cellular activity may be a hematological and / or solid tumor selected from, for example, esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, liver cancer, kidney cancer, urothelial cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, or thyroid cancer.

[0200] In some embodiments, the disease is preferably a B7H3-associated cancer disease.

[0201] In some embodiments, the disease is preferably a HER2-associated cancer disease.

[0202] In some embodiments, the disease is preferably a TROP2-associated cancer disease.

[0203] In some embodiments, the disease is preferably an EGFR-associated cancer disease.

[0204] In some embodiments, the disease is preferably a solid tumor.

[0205] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the experimental steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all common steps widely used in the art. At the same time, the definitions and explanations of related terms are provided below to better understand this specification.

[0206] Examples of the term "pharmaceutically acceptable salts" as used herein are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including, but not limited to, formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarate, α-glycerophosphate, alkylsulfonates or arylsulfonates, preferably the alkylsulfonates being methylsulfonates or ethylsulfonates and the arylsulfonates being benzenesulfonates or p-toluenesulfonates. Suitable inorganic salts may also be formed, including, but not limited to, hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates and carbonates, sulfates or phosphates.

[0207] Pharmaceutically acceptable salts can be obtained by standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with an appropriate acid to provide a pharma-ceutically acceptable anion.

[0208] In this specification, the pharmaceutical auxiliary refers to excipients and additives used in the manufacture and preparation of pharmaceuticals, and refers to substances contained in drug preparations whose safety has been reasonably evaluated other than the active ingredient. Pharmaceutical auxiliary acts as a molding and carrier, improves stability, and also has important functions such as solubilization, auxiliary dissolution, sustained release and release control, and is an important component that may affect the quality, safety and efficacy of drugs. According to their origin, they may be divided into natural products, semi-synthetic products and fully synthetic products. According to their effects and uses, they may be classified into solvents, propellants, solubilizers, dissolution aids, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, clathrates, moisturizers, absorbents, diluents, flocculants and deflocculants, filter aids, release blockers, etc. According to the administration route, they may be classified into oral administration, injection, mucosal administration, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical auxiliary may be used in pharmaceutical preparations with different administration routes and have different effects and uses.

[0209] The pharmaceutical composition can be prepared into various suitable dosage forms depending on the route of administration. For example, tablets, capsules, granules, oral liquids, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye drops, pills, implants, aerosols, powdered aerosols, sprays, etc. Here, the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or a pharma- ceutically acceptable salt or complex thereof, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, and particularly preferably 1 to 250 mg.

[0210] The pharmaceutical composition can be administered as an injection, such as an injection, a sterile powder for injection, or a concentrated solution for injection. Here, usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspension media.

[0211] The term "treatment" as used herein generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it prevents, completely or partially, a disease or its symptoms, and / or therapeutic, in that it stabilizes or cures, partially or completely, the disease and / or side effects of the disease. As used herein, "treatment" includes any treatment of a disease in a patient, including (a) preventing a disease or condition in a patient susceptible to the disease or condition but not yet diagnosed, (b) inhibiting the symptoms of the disease, i.e., preventing its onset, or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or condition.

[0212] In this disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) with a disease (e.g., a disease described herein) or normal individuals. In this disclosure, the term "non-human animal" includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals (e.g., non-human primates, pets and / or farm animals (e.g., sheep, dogs, cats, dairy cows, pigs, etc.)).

[0213] In this disclosure, the term "effective dose" refers to that amount of a compound which, upon administration, relieves to some extent one or more of the symptoms of the condition being treated.

[0214] In the present disclosure, the term "antibody-eribulin or derivative thereof conjugate" refers to a substance obtained by linking eribulin or a derivative thereof to a targeting moiety. In some embodiments of the present disclosure, eribulin or a derivative thereof is linked to the targeting moiety by a linker. The linker can be cleaved under a specific environment or action, thereby separating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit such as a peptide or disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety by a covalent bond that can be cleaved under a specific environment or action, thereby separating the bioactive molecule from the targeting moiety.

[0215] In this disclosure, the term "linker" refers to a fragment that connects a bioactive molecule (a drug molecule) to a targeting moiety.

[0216] In this disclosure, the term "targeting moiety" refers to a moiety in a complex that can specifically bind to a target (or a portion of a target) on a cell surface. The interaction of the targeting moiety with the target allows the complex to be delivered to a specific population of cells.

[0217] In this disclosure, when the targeting moiety in the conjugate is an antibody, the conjugate may be referred to as a "drug-antibody conjugate."

[0218] In the present disclosure, an antibody or antigen-binding fragment thereof includes a derivatized antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof bearing a sulfhydryl, in which the derivatization allows the antibody to have a group or ability to react with a drug-linker conjugate. The sulfhydryl-SH can be derivatized by opening a disulfide bond (e.g., reduction with the reducing agent TCEP).

[0219] In the present disclosure, the term "antibody" is used in the broadest sense and includes complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, so long as they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" are used interchangeably. As used herein, "antibody molecule" or "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody in its broadest sense not only includes complete antibody molecules, but also antibody fragments and variants (including derivatives) of the antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as an antigen-binding fragment, "antibody molecule" or "antibody" refers to a complete antibody molecule or a full-length antibody. As used herein, the term antibody molecule includes, but is not limited to, for example, single chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, Fvs linked by disulfide bonds (sdFv), Fvs, and complete or full-length antibodies. The term "single chain Fv" or "scFv" refers to a polypeptide comprising an antibody VL domain linked to an antibody VH domain. For example, an antibody that immunospecifically binds to B7H3 may cross-react with other antigens. Preferably, an antibody that immunospecifically binds to B7H3 does not cross-react with other antigens. An antibody that immunospecifically binds to B7H3 can be identified, for example, by immunoassays or other methods known to those of skill in the art. A "complete" or "full-length" antibody refers to a protein comprising two heavy chains (H) and two light chains (L) linked together by disulfide bonds, the protein comprising: (1) a heavy chain, a variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains CH1, CH2, CH3; and (2) a light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising one domain CL.Antibodies of the present disclosure include, but are not limited to, monoclonal antibodies, multispecific antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, the anti-Id antibodies of the present disclosure), and epitope-binding fragments of any of the above antibodies. Immunoglobulin molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin. Preferably, antibodies of the present disclosure are comprised of a VH domain, a VH CDR (often referred to herein as HCDR), a VL domain, or a VL CDR (often referred to herein as LCDR) having any of the amino acid sequences set forth in the Table of Sequences and Specific Information thereof, or fragments or variants thereof.

[0220] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical except for a small number of natural variations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies contain different antibodies for different determinants (epitopes). In addition to specificity, an advantage of monoclonal antibodies is that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" as used herein means that the antibody has the character of being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by any particular method.

[0221] In some embodiments of the present disclosure, monoclonal antibodies further include, inter alia, chimeric antibodies, i.e., a portion of the heavy and / or light chain is identical or homologous to one type, class or subclass of antibody, and the remaining portion is identical or homologous to another type, class or subclass of antibody, so long as it possesses the desired biological activity (see, e.g., US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies that contain variable region antigen-binding sequences derived from a non-human primate (e.g., an ancient monkey, chimpanzee, etc.) and human constant region sequences.

[0222] The term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single chain antibody molecules.

[0223] The term "bispecific antibody", also called "bifunctional antibody complex", refers to a complex formed through binding arms of a first antibody (fragment) and a second antibody (fragment), which complex retains the activity of each antibody and has bifunctional and bispecific properties.

[0224] The term "multispecific antibody" includes, for example, triabodies, which are antibodies with three different antigen-binding specificities, and tetrabodies, which are antibodies with four different antigen-binding specificities.

[0225] The term "intact antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2 and CH3). The constant regions may be native sequences (e.g., human native constant region sequences) or amino acid sequence variants thereof. The intact antibody is preferably a complete antibody with one or more effector functions.

[0226] The term "probody" refers to a modified antibody comprising an antibody or antibody fragment that specifically binds to its target and is capable of being conjugated to a masking group, where the masking group means that the cleavage constant for the binding ability of the antibody or antibody fragment to its target is at least 100-fold, 1000-fold, or 10000-fold greater than the cleavage constant for the binding ability of the antibody or antibody fragment not conjugated to the masking group to its target.

[0227] In this disclosure, a "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that contains minimal non-human immunoglobulin sequence. Most humanized antibodies are human recipient immunoglobulins whose hypervariable region residues can be replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Furthermore, humanized antibodies may contain additional residues that are not present in the recipient antibody or in the donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies generally contain at least one variable region, usually two, in which all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, and the FRs are complete or nearly complete sequences of a human immunoglobulin. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For further details, see, e.g., Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

[0228] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and tertiary structures of the different classes of immunoglobulins are known in the art.

[0229] In the present disclosure, the amino acid substitutions in the antibody are primarily, but not limited to, L-amino acids. In some embodiments, the antibody peptide chain may contain one or more D-amino acids. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0230] The monoclonal antibodies used in the present disclosure can be produced by several methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by the hybridoma method using many species (including mouse, hamster, rat and human cells) (see, e.g., Kohler et al., 1975, Nature, 256:495), or can be produced by recombinant DNA techniques (see, e.g., US 4,816,567), or can be isolated from phage antibody libraries (see, e.g., Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0231] In some embodiments, the Tb is a targeting moiety, such as a ligand, a protein, a polypeptide, a non-proteinaceous reagent (eg, a sugar, RNA or DNA), an antibody analog, or the like.

[0232] In this disclosure, the term "targeting moiety" refers to a moiety in a complex that can specifically bind to a target (or a portion of a target) on a cell surface. Through interaction of the targeting moiety with the target, the complex may be delivered to a specific population of cells.

[0233] In the present disclosure, when Tb in a conjugate is a targeting moiety, the conjugate may be referred to as an "antibody-eribulin or a derivative thereof conjugate."

[0234] In some embodiments of the present disclosure, the targeting moiety is trastuzumab or pertuzumab, an anti-Her2 monoclonal antibody whose amino acid sequence is known to those skilled in the art and whose outline sequence can be found, for example, in CN103319599, and whose terminal Lys is easily deleted without affecting biological activity (see Dick, LW et al., Biotechnol. Bioeng., 100:1132-1143).

[0235] In some embodiments of the present disclosure, the targeting moiety anti-Trop-2 antibody is described in US Patent No. 7,517,964 as RS7 (i.e., Sacituzumab of the present disclosure) and in US2012 / 0237518 as hRS7 (i.e., Sacituzumab of the present disclosure). Anti-Trop-2 antibodies that can be used in the present disclosure can also be obtained by designing, constructing and screening vectors as disclosed in CN103476941A and constructing antibody libraries displaying antibodies, or by screening the G-MAB® library of Sorrento Therapeutics, Inc.

[0236] In the present disclosure, ErbB2 and Her2 / neu may be used interchangeably, both referring to the native sequence human Her2 protein (Genebank Accession No.: X03363, see e.g., Semba et al., 1985, PNAS, 82:6497-6501; and Yamamoto et al., 1986, Nature, 319:230-234) and its functional derivatives, e.g., amino acid sequence variants. ErbB2 refers to the gene encoding human Her2, and neu refers to the gene encoding rat p185neu. In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, kidney cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells, or liver cancer cells.

[0237] In the present disclosure, Trop-2 or TROP2 refers to human trophoblast cell-surface antigens 2, also referred to as TACSTD2, M1S1, GA733-1, EGP-1, which is a cell surface receptor expressed by many human tumor (e.g., breast, colon, lung, pancreatic, ovarian, prostate, cervical) cells. In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing the TROP2 receptor, such as breast cancer cells, colon cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells, or cervical cancer cells.

[0238] In some embodiments of the present disclosure, the targeting moiety is an anti-B7H3 monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the anti-B7H3 antibody includes all prior art anti-B7H3 antibodies, such as CN112521512, WO2021027674, WO2021021543, WO2021006619, CN111662384, CN111454357, WO2020151384, WO2020140094, WO2020103100, WO2020102779, WO2020063673, WO2020047257, WO2020041626, CN110684790, CN110 642948, WO2019225787, WO2019226017, US20190338030, CN110305213, WO2018209346, WO2018177393, US9150656, WO2016106004, WO2016044383, WO2016033225, WO2015181267, US20120294796, WO2011109400, CN101104639, WO2004093894, WO2002010187, and WO2001018021.

[0239] In some embodiments of the present disclosure, the anti-B7H3 antibody is selected from the antibody numbered M30-H1-L4 in CN103687945B and the antibody numbered mAb-C-DUBA in CN109069633A. In some embodiments of the present disclosure, the anti-B7H3 antibody is selected from 1D1 or 2E3.

[0240] In this specification, unless otherwise specified, the expressions "each independently selected ..." and "each independently selected ..." used throughout this specification may be used interchangeably and should be understood in a broad sense, and may mean that specific options represented by the same or different symbols in different groups do not affect each other, and may also mean that specific options represented by the same or different symbols in the same group do not affect each other.

[0241] AA 1The structure of the amino acid residue represented by [ka] In the case where r is 0, those skilled in the art can 1 The structure of the amino acid residue represented by [ka] It is possible to understand that the

[0242] AA 1 The structure of the amino acid residue represented by [ka] In R a and R b When they form a 4- to 10-membered heterocyclic ring together with the carbon atom to which they are attached, the 4- to 10-membered heterocyclic ring may optionally be joined by one or more R 0 wherein the term "the 4- to 10-membered heterocycle is optionally substituted with one or more R 0 "substituted by" means that the 4-10 membered heterocycle may be unsubstituted or may be substituted by one or more R 0 and each of the R 0 In each R 0 The definitions of may be the same or different. Other similar definitions can be understood by referring to the above.

[0243] At various places in the present specification, substituents of the compounds of the present disclosure are disclosed according to radical types or ranges. In particular, the present disclosure includes each independent subcombination of each member of these radical types and ranges. For example, the term "C 1-6 "Alkyl" specifically refers to methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, each of which is disclosed independently.

[0244] As used herein, the term “C 1-6 "Alkyl" refers to a straight or branched alkyl group containing 1 to 6 carbon atoms, e.g., "C 1-3 Alkyl" or "C 1-4 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0245] As used herein, the term “C 1-4 "Alkyl" refers to a straight or branched alkyl group containing 1 to 4 carbon atoms, e.g., "C 1-3 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0246] As used herein, the term “C 2-6 "Alkenyl" refers to a straight, branched or cyclic alkenyl containing at least one double bond and 2 to 6 carbon atoms, e.g., "C 2-4 Examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0247] As used herein, the term “C 2-6 "Alkynyl" refers to a straight or branched alkynyl containing at least one triple bond and 2 to 6 carbon atoms, e.g., "C 2-4Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0248] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0249] As used herein, the term "3- to 6-membered cycloalkyl" or "C 3-6 "Cycloalkyl" refers to a saturated cyclic alkyl containing from 3 to 6 carbon atoms, including cyclopropane (cyclopropyl), cyclobutane (cyclobutyl), cyclopentane (cyclopentyl), and cyclohexyl.

[0250] As used herein, the term "3- to 7-membered carbocycloalkyl" or "C 3-7 "Cycloalkyl" refers to a saturated cyclic alkyl group containing from 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0251] As used herein, the term "3- to 8-membered carbocyclic ring" refers to a saturated, unsaturated or partially unsaturated carbocyclic ring containing 3 to 8 carbon atoms. Specific examples include, but are not limited to, cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexane (i.e., cyclohexyl).

[0252] As used herein, the term “C 1-6 "Alkoxy" refers to an alkyl, as defined above, attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexyloxy, and the like.

[0253] As used herein, the term “C 1-4"Alkoxy" refers to an alkyl, as defined above, attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0254] As used herein, the term "4-10 membered heterocyclyl" refers to a cyclic group containing 4 to 10 ring atoms, in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "4-6 membered heterocyclyl" refers to a cyclic group containing 4 to 6 ring atoms, in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, a ring atom in the cyclic structure (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) may be oxidized. Examples of "4- to 8-membered heterocyclyl" include "4- to 8-membered nitrogen-containing heterocyclyl", "4- to 8-membered oxygen-containing heterocyclyl", "4- to 7-membered heterocyclyl", "4- to 7-membered oxygen-containing heterocyclyl", "4- to 7-membered heterocyclyl", "4- to 6-membered heterocyclyl", "5- to 7-membered heterocyclyl", "5- to 6-membered heterocyclyl", and "5- to 6-membered nitrogen-containing heterocyclyl", including, but not limited to, oxetanyl, pyrrolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like.

[0255] As used herein, the term "4- to 10-membered heterocyclic ring" refers to a ring containing 4 to 10 ring atoms (at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom). The term "5- to 6-membered heterocyclic ring" refers to a ring containing 5 to 6 ring atoms (at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom), including, but not limited to, rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, etc.

[0256] As used herein, the term "3-8 heterocyclic ring" refers to a ring containing 3 to 8 ring atoms, of which at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom.

[0257] In the present specification, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon group such as 6- to 10-membered aryl, 5- to 8-membered aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, etc. The "6- to 10-membered aryl" refers to an aryl containing 6 to 10 ring atoms. 6-10 "Aryl" refers to an aryl containing 6 to 10 carbon atoms.

[0258] As used herein, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (e.g., carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5-10 membered heteroaryl, 5-6 membered heteroaryl, 5-10 membered nitrogen-containing heteroaryl, 6-10 membered oxygen-containing heteroaryl, 6-8 membered nitrogen-containing heteroaryl, 5-8 membered oxygen-containing heteroaryl, and the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, and the like. Examples of aryl include, but are not limited to, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxacyclohexadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0259] As used herein, a bond in a structural formula represented by a wavy line "~~" is intended to indicate that the structure represents either the cis-isomer or the trans-isomer, or a mixture of cis- and trans-isomers in any ratio.

[0260] Each of the above preferred conditions can be arbitrarily combined to obtain each preferred embodiment of the present invention, without violating the ordinary skill in the art.

[0261] The reagents and materials used in the present invention are commercially available.

[0262] The positive advances of the present invention are: The antibody-eribulin or its derivative of the present disclosure is (1) High antitumor activity, which can kill tumor cells more effectively, and has a better antitumor effect in vivo or in vitro than the antibody-eribulin or its derivative linked to the same small molecule drug in the prior art; (2) The high stability of the bioactive molecules in circulation reduces the shedding of non-target bioactive molecules in non-target cells, increases the effective release of the bioactive molecules in cells, and allows the bioactive molecules to have a higher intratumoral and blood concentration ratio, resulting in a higher therapeutic index; (3) By adding a basic nitrogen atom to the linker, the exposure of the entire antibody-eribulin or its derivative molecule in the relatively acidic tumor environment is increased, improving targeting of tumor tissue and increasing the concentration ratio of the biologically active molecule in the tumor and in the blood, resulting in a higher therapeutic index. At least one of the following can be achieved.

[0263] Sequence and its specific information: [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Brief description of the drawings]

[0264] [Figure 1A] ELISA binding detection of anti-B7H3 antibody 1D1-01 and human B7H3-4 IgG-His protein. [Figure 1B] ELISA binding detection of anti-B7H3 antibody 2E3-02 and human B7H3-4 IgG-His protein. [Figure 2A] ELISA binding detection of anti-B7H3 antibody 1D1-01 and monkey B7H3-4 IgG-His protein. [Figure 2B] ELISA binding detection of anti-B7H3 antibody 2E3-02 and monkey B7H3-4 IgG-His protein. [Figure 3A] Anti-B7H3 antibody 1D1-01 and flow cytometric detection of MCF-7 tumor cells. [Figure 3B] Anti-B7H3 antibody 2E3-02 and flow cytometric detection of MCF-7 tumor cells. [Figure 4A] Anti-B7H3 antibody 1D1-01 and flow cytometric detection of A549 tumor cells. [Figure 4B] Anti-B7H3 antibody 2E3-02 and flow cytometric detection of A549 tumor cells. [Diagram 5] Anti-B7H3 antibody 2E3-02 and flow cytometric detection of PC-3 tumor cells. [Figure 6A] Endocytosis detection of tumor cell A549 against candidate antibody 1D1-01. [Figure 6B] Endocytosis detection of tumor cell A549 for candidate antibody 2E3-02. [Figure 7A] Detection of the inhibitory activity of B7H3-ADC-01 in SW480 cells. [Figure 7B]Detection of the inhibitory activity of B7H3-ADC-01 in A375 cells. [Figure 7C] Detection of the inhibitory activity of B7H3-ADC-01 in A549 cells. [Figure 7D] Detection of the inhibitory activity of B7H3-ADC-01 in HT29 cells. [Figure 7E] Detection of the inhibitory activity of HER2-ADC-04 in H358 cells. [Figure 8A] Detection of tumor growth inhibition of B7H3-ADC-01 in NCI-HT29 xenograft tumor model. [Figure 8B] This shows the change in body weight of each mouse in the NCI-HT29 tumor transplant model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0265] The present invention will be further described below in the form of examples, but the present invention is not limited to the scope of the examples. In the following examples, the experimental methods for which no specific conditions are described are selected according to the usual methods and conditions or product instructions.

[0266] The abbreviations in this disclosure have the following meanings: [Table 8]

[0267] The structures of the compounds described in the following examples can be determined by nuclear magnetic resonance ( 1 The molecular weight is determined by 1 H NMR or mass spectrometry (MS).

[0268] Nuclear magnetic resonance ( 1 The measurement apparatus used for H NMR was a Bruker 400 MHz nuclear magnetic resonance apparatus, the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or hexadeuterated dimethyl sulfoxide (DMSO-d6), and the internal standard was tetramethylsilane (TMS).

[0269] The abbreviations for nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0270] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0271] The mass spectrometry (MS) detection device used was an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0272] 1. Synthesis of intermediates used in the synthesis of biologically active molecules and "drug-linker compounds" Example 1 (A1): N-2-aminoacetyl-eribulin [ka]

[0273] Step 1: N-2-(tert-butoxycarbonylamino)acetyl-eribulin Compound (A1.1) (21 mg) in dichloromethane (1 ml) was added with DIPEA (100 ul) and HBTU (45 mg), and the mixture was stirred at room temperature for 20 minutes to react, and then eribulin (A1.2, 73 mg) was added to the reaction solution. The reaction solution was stirred at room temperature for 10 hours to react, and then diluted with ethyl acetate (50 ml), washed with water (20 ml x 3) and saturated aqueous sodium chloride solution (20 ml), dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure, and compound A1.3 was obtained by separation by silica gel column chromatography (ethyl acetate: petroleum ether). ESI-MS (m / z): 887.5 [M+H] + .

[0274] Step 2: N-2-aminoacetyl-eribulin N-2-(tert-butoxycarbonylamino)acetyl-eribulin (A1.3, 80 mg) was added with dichloromethane (2 ml) and TFA (1 ml). The mixture was stirred for 10 minutes, then the low boiling point components were removed under reduced pressure, the crude product was dissolved in ethyl acetate (50 ml), saturated aqueous sodium bicarbonate solution (15 ml) was added and mixed thoroughly, the organic phase was washed with water (20 ml x 3) and saturated aqueous sodium chloride solution (20 ml), dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain compound N-2-aminoacetyl-eribulin (A1). ESI-MS (m / z): 787.5 [M+H] + .

[0275] B. Synthesis of fragment intermediates containing coupling linker molecules Example 2 (B1): N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (Compound B1) [ka]

[0276] 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoic acid (920 mg) and N-hydroxysuccinimide (537 mg) were dissolved in dichloromethane (50 mL) and then dicyclohexylcarbodiimide (963 mg) was added. The reaction solution was stirred at room temperature for 1 hour, then the dichloromethane was removed under reduced pressure and the residue was dissolved in N,N-dimethylformamide (50 mL) and then N 2 -L-Baryl-N 6 -(Boc)-L-lysine (1.5 g) was added, and the reaction solution was stirred at room temperature for 16 hours to react. The reaction solution was poured into 200 mL of water, and the pH was adjusted to 11 with saturated sodium bicarbonate solution. The above aqueous solution was extracted twice with ethyl acetate, the organic phase was discarded, the aqueous phase was adjusted to pH 4-5 with citric acid, and extracted three times with ethyl acetate (100 ml × 3). The organic phase was washed with saturated saline (100 ml), dried over anhydrous sodium sulfate, suction filtered, and spin evaporated to obtain 2.8 g of crude product. The crude product was separated and purified by flash chromatography (DCM:MeOH = 30:1) to obtain the target compound N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (1.0 g) was obtained.

[0277] ESI-MS (m / z): 564.3 [M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.68 (s, 2H), 8.12 (d, J = 7.3 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 6.76 (s, 1H), 4.27 - 4.19 (m, 1H), 4.11 (d, J = 4.9 Hz, 1H), 2.88 (d, J = 7.9 Hz, 3H), 2.73 (s, 1H), 2.59 (s, 1H), 2.52 (s, 4H), 2.40 - 2.24 (m, 3H), 2.03 - 1.88 (m, 2H), 1.77 (d, J = 3.2 Hz, 2H), 1.68 (d, J = 7.0 Hz, 1H), 1.60 - 1.49 (m, 1H), 1.36 (s, 15H), 0.85 (dd, J = 14.8, 6.7 Hz, 7H).

[0278] Example 3 (B2): N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (Compound B2) [ka]

[0279] To a mixed solution of compound B1 (260 mg) in tetrahydrofuran (3 ml) and water (3 ml), potassium peroxymonosulfate (1.414 g) was added and the mixture was stirred at room temperature for 1 hour to react. The reaction solution was filtered, and the filtrate was purified using a C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5% to 60%) to obtain the target compound N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (compound B2) (120 mg) was obtained.

[0280] LCMS (ESI) [M+H] + : 596;1 H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 9.13 (s, 2H), 8.14 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 6.77 (s, 1H), 4.24 (t, J = 7.7 Hz, 1H), 4.12 (br s, 1H), 3.41 (s, 3H), 2.89 (d, J = 6.0 Hz, 2H), 2.43 - 2.29 (m, 2H), 2.03 - 1.93 (m, 2H), 1.82 (br s, 2H), 1.63 - 1.60 (m, 4H), 1.37 (s, 12H), 0.88 - 0.83 (m, 6H).

[0281] Example 4 (B3): N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valyl)-L-lysine (Compound B3) [ka]

[0282] Using the same method and reaction conditions as in Example B1, the target product N was obtained using the known compound shown in the reaction formula as the raw material. 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valyl)-L-lysine (compound B3) was obtained. ESI-MS (m / z): 611 [M+H] + .

[0283] Example 5 (B4): N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (Compound B4) [ka]

[0284] Step 1: 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid At room temperature, 2-methylthio-5-ethynylpyrimidine (1.5 g) and methyl 6-azidohexanoate (1.71 g) were dissolved in a mixed solvent of tert-butanol and water (20 ml / 25 ml), and sodium vitamin C (5.7 g) and cuprous bromide (2.9 g) were added and reacted with stirring for 10 hours. Ethyl acetate (200 ml) was added to the reaction system, washed with water (100 ml x 5), dried, and then the organic solvent was removed. Methyl 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoate (2.8 g) was obtained by silica gel column chromatography. ESI-MS (m / z): 321.9 [M+H] + The above product was dissolved in tetrahydrofuran (100ml), lithium hydroxide (1M, 20ml) was added, the mixture was stirred for 3 hours, then hydrochloric acid was added to the reaction solution to adjust the pH to 2, followed by addition of ethyl acetate (200ml), washing with water (100ml x 3), drying, and removing the organic solvent to obtain the target product 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid. ESI-MS (m / z): 308.4 [M+H] + .

[0285] Step 2:N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine Compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (370 mg) and N-hydroxysuccinimide (152 mg) were dissolved in dichloromethane (5 ml) and stirred, dicycloethylcarbodiimide (273 mg) was further added, and the reaction solution was stirred at room temperature for 1 hour. 10 ml of water was added to the reaction solution, and it was extracted with ethyl acetate (20 ml x 2). The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in N,N-dimethylformamide (10 ml) and stirred to obtain compound N. 2 -L-Baryl-N 6 -(Boc)-L-lysine was added, and the reaction solution was reacted at room temperature for 16 hours. Citric acid was slowly added to the reaction solution to adjust the pH to about 5, water was added, and the solution was extracted with ethyl acetate (3×10 ml). The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate, and the target product N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (compound B4, 400 mg) was obtained.

[0286] ESI-MS (m / z): 635.3 [M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 9.06 (s, 2H), 9.06 (s, 1H), 8.70 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 6.76 (t, J = 5.6 Hz, 1H), 4.41 (t, J = 7.0 Hz, 2H), 4.19 (dd, J = 8.9, 7.0 Hz, 1H), 4.14 - 4.05 (m, 1H), 2.92 - 2.85 (m, 2H), 2.56 (s, 3H), 2.24 - 2.10 (m, 2H), 1.97 - 1.81 (m, 3H), 1.71 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.36 (s, 9H), 1.28 - 1.22 (m, 6H), 0.88 - 0.76 (m, 6H).

[0287] Example 6 (B5): N 6 -(tert-butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazanonatriacont-38-ynoyl)-L-valyl)-L-lysine (B5) [ka]

[0288] Step 1: Compound B5.1 (336 mg) was dissolved in dichloromethane (5 ml), N-hydroxysuccinimide (98 mg, 0.85 mmol) and dicyclohexylcarbodiimide (175 mg, 0.85 mmol) were added, and the mixture was stirred at room temperature for 1 hour to react. 2 -L-Baryl-N 6-(Boc)-L-lysine (230 mg) was added to the reaction solution, and the mixture was stirred at room temperature overnight to react. The mixture was concentrated to remove the solvent to obtain a crude product, which was then separated and purified using a C18 reversed-phase column (acetonitrile / 0.01% FA aqueous solution: 5%-50%) to obtain the colorless oily target compound (B5.2, 290 mg).

[0289] LCMS (ESI) [M+H] + = 882.3.

[0290] Step 2: Compound B5.2 (300 mg) was dissolved in anhydrous methanol (5 mL), Pd / C (10%, 60 mg) was added, and the mixture was purged with hydrogen gas three times. The mixture was stirred at room temperature overnight, filtered by suction, and concentrated to give the target compound B5.3 (293 mg), which was a colorless oil.

[0291] LCMS (ESI) [M+H] + :856.1.

[0292] Step 3: 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoic acid (84 mg) was dissolved in N,N-dimethylformamide (3 mL), and then N,N-diisopropylethylamine (61 mg) and HATU (108 mg) were added in sequence. The reaction solution was stirred at room temperature for 20 minutes, and then compound B5.3 (196 mg) was added and reacted by stirring at 40°C for 3 hours. The crude product was separated and purified by a reversed-phase C18 column (acetonitrile / 0.01% FA aqueous solution: 5%-65%) to obtain the target compound N. 6 -(tert-butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazanonatriacont-38-ynoyl)-L-valyl)-L-lysine (B5) (170 mg) was obtained.

[0293] LCMS (ESI) [M+H] + :1074.0; 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 2H), 8.23 ​​(t, J = 5.7 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.43 (d, J = 6.5 Hz, 1H), 6.69 (s, 1H), 4.09 (dd, J = 8.8, 6.5 Hz, 1H), 4.02 (s, 2H), 3.98 (d, J = 2.9 Hz, 2H), 3.74 - 3.70 (m, 1H), 3.50 (s, 30H), 3.45 (d, J = 6.2 Hz, 2H), 3.39 (d, J = 5.8 Hz, 2H), 3.26 (d, J = 6.1 Hz, 2H), 3.20 (q, J = 5.9 Hz, 2H), 2.81 (t, J = 6.6 Hz, 2H), 2.52 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 2.12 - 2.06 (m, 1H), 1.79 - 1.74 (m, 2H), 1.67 - 1.59 (m, 1H), 1.50 (d, J = 5.3 Hz, 1H), 1.36 (s, 9H), 1.24 (s, 2H), 1.20 - 1.09 (m, 2H), 0.85 (t, J = 6.3 Hz, 6H).

[0294] Example 7 (B6): N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [ka]

[0295] Step 1: A solution of hydrogen chloride in dioxane (100 ml) was added to compound B6.1 (10.0 g) and reacted at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the target compound B6.2 (8.0 g) as a white solid.

[0296] LCMS (ESI) [M+H] + :380.1. 1 H NMR (400 MHz, DMSO) δ 8.18 (d, J = 7.4 Hz, 1H), 7.40 - 7.30 (m, 5H), 7.26 (d, J = 8.8 Hz, 1H), 5.08 - 4.99 (m, 2H), 4.23 - 4.11 (m, 1H), 3.94 - 3.88 (m, 1H), 2.78 - 2.73 (m, 2H), 2.03 - 1.94 (m, 1H), 1.77 - 1.51 (m, 4H), 1.44 - 1.31 (m, 2H), 0.87 (dd, J = 17.3, 6.6 Hz, 6H).

[0297] Step 2: Compound B6.2 (3.0 g) and sodium acetate (1.90 g) were dissolved in methanol (100 ml) solution and reacted at room temperature for 10 minutes, paraformaldehyde (2.8 g) was added to the reaction solution, and the reaction was stirred at room temperature for 30 minutes, and sodium cyanoborohydride (1.0 g) was added to the reaction solution, and the reaction system was stirred at room temperature for 16 hours. After filtering the reaction solution, the filtrate was separated and purified using a reverse phase C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5% to 55%) to obtain the target compound B6.3 (1.70 g).

[0298] LCMS (ESI) [M+H] + :408.1; 1 H NMR (400 MHz, DMSO) δ 7.86 (d, J = 7.3 Hz, 1H), 7.40 - 7.26 (m, 6H), 5.03 (s, 2H), 4.08 - 4.06 (m, 1H), 3.90 - 3.85 (m, 1H), 2.50 - 2.45 (m, 2H), 2.35 (s, 6H), 2.05 - 1.93 (m, 1H), 1.73 - 1.55 (m, 2H), 1.51 - 1.40 (m, 2H), 1.33 - 1.22 (m, 2H), 0.85 (dd, J = 16.5, 6.8Hz, 6H).

[0299] Step 3: Compound B6.3 (1.6 g) was dissolved in methanol (80 ml) at room temperature, and then Pd / C (10%, 0.16 g) was added to the reaction solution, and the mixture was stirred under hydrogen gas at room temperature for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B6.4 (680 mg). LCMS (ESI) [M+H] + :274.2;

[0300] Step 4: 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoic acid (944 mg) was dissolved in N,N-dimethylformamide (30 mL), and then N,N-diisopropylethylamine (1.3 g) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1.8 g) were added in sequence. The reaction solution was stirred at room temperature for 20 minutes, and then compound B6.4 (1.1 g) was added and reacted at 40 °C for 3 hours with stirring. The crude product was separated and purified by reverse phase C18 column (acetonitrile / 0.01% formic acid aqueous solution: 5%-65%) to obtain the target compound N as a white solid. 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine B6 (1.2 g) was obtained.

[0301] LCMS (ESI) [M+H] + :492.1; 1H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 8.00 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 4.20 (t, 1H), 4.08 (dd, J = 12.7, 7.7 Hz, 1H), 2.47 - 2.40 (m, 4H), 2.37 - 2.31 (m, 2H), 2.30 (s, 6H), 2.01 - 1.92 (m, 1H), 1.82 - 1.73 (m, 2H), 1.71 - 1.56 (m, 2H), 1.49 - 1.37 (m, 2H), 1.33 - 1.23 (m, 2H), 0.88 - 0.82 (m, 6H).

[0302] Example 8 (B7): N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (B7) [ka]

[0303] 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg), compound B6.4 (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 ml). Then, 1-hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were added, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound N. 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (B7, white solid, 327 mg) was obtained.

[0304] LCMS (ESI) [M+H] + :524.4. 1 H NMR (400 MHz, ) δ 9.13 (s, 2H), 7.95 (t, J = 8.8 Hz, 2H), 4.21 (dd, J = 8.8, 6.9 Hz, 1H), 4.08 - 4.03 (m, 1H), 3.41 (s, 3H), 2.55 (t, J = 7.0 Hz, 2H), 2.42 - 2.32 (m, 4H), 2.27 (s, 6H), 1.98 (dd, J = 13.6, 6.8 Hz, 1H), 1.86 - 1.77 (m, 2H), 1.74 - 1.55 (m, 2H), 1.47 - 1.37 (m, 2H), 1.31 - 1.23 (m, 2H), 0.85 (dd, J = 12.8, 6.8 Hz, 6H).

[0305] Example 9 (B8): N 2 -(tert-butoxycarbonyl)-N 6 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [ka]

[0306] Step 1: Compound B8.2 (3g) was dissolved in dichloromethane (30ml), DIPEA (4ml) was added, then compound B8.1 (3.48g) was added, and the mixture was stirred at room temperature for 20 hours to react. Ethyl acetate (200ml) was added to the reaction solution, and the mixture was washed successively with hydrochloric acid (0.1M, 30ml x 3), water (30ml x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain target mixture B8.3 (4.7g).

[0307] Step 2: Compound B8.3 (4.7 g) was dissolved in methanol (80 ml) at room temperature, and then Pd / C (10%, 0.6 g) was added to the reaction solution, and the mixture was stirred under hydrogen gas at room temperature for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B8.4 (3.4 g). LCMS (ESI) [M+H] + :346.2.

[0308] Step 3: 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (2.68 g) was dissolved in N,N-dimethylformamide (50 ml), then triethylamine (3 ml) and HBTU (3.8 g) were added, and the mixture was stirred at room temperature for 10 minutes, after which ethyl acetate (200 ml) was added, and the mixture was washed with saturated sodium bicarbonate (20 ml x 2), hydrochloric acid (0.1 M, 50 ml x 2), water (50 ml x 2), and dried over anhydrous sodium sulfate. The crude intermediate was obtained by dissolving the crude intermediate in DMF (30 ml), adding DIPEA (1.6 ml), and then adding B8.4 (3.4 g). The reaction solution was stirred at room temperature for 3 hours, after which ethyl acetate (200 ml) was added, washed with hydrochloric acid (0.1 M, 50 ml x 2), water (50 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude intermediate. The crude intermediate was separated and purified by silica gel column chromatography to obtain the target compound N 2 -(tert-butoxycarbonyl)-N 6 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (B8, white solid, 3.8 g) was obtained.

[0309] LCMS (ESI) [M+H] + :596.4.

[0310] Example 10 (B9): N 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine [ka]

[0311] Step 1: Compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (1.0 g, 3.3 mmol) was dissolved in a mixture of tetrahydrofuran and water (40 mL, 3:1), and potassium peroxymonosulfate (10.0 g, 16.3 mmol) was added. The reaction was stirred at room temperature for 3 hours, and the completion of the reaction was detected by LCMS. The reaction solution was filtered, the cake was washed with DMSO, and the filtrate was combined. Purification by reverse phase (C18, acetonitrile: 0.1% formic acid = 5% ~ 55%) gave 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (750 mg, yield: 67.9%) as a white solid.

[0312] LCMS (ESI) [M+H] + = 340.1;

[0313] 1 H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 2H), 8.95 (s, 1H), 4.49 (t, J = 7.0 Hz, 2H), 3.45 (s, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.95 - 1.84 (m, 2H), 1.61 - 1.50 (m, 2H), 1.36 - 1.26 (m, 2H).

[0314] Step 2: Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (300 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (6 mL), HATU (337 mg, 0.88 mmol) and DIPEA (286 mg, 2.21 mmol) were added, and the mixture was stirred at room temperature for 30 minutes to react. Then, dipeptide B6.4 (243 mg, 0.88 mmol) was added, and the mixture was stirred at room temperature for 2 hours to react. The completion of the reaction was detected by LCMS. The reaction solution was separated and purified by a C18 column (acetonitrile / 0.01% FA aqueous solution: 5%-50%) to obtain the target compound N 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (300 mg, yield: 57%) was obtained as a white solid.

[0315] LCMS (ESI) [M+H] + = 595.5;

[0316] 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 9.01 (s, 1H), 7.85 (br s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 4.48 (t, J = 6.8 Hz, 2H), 4.17 - 4.15 (m, 1H), 4.02 (br s, 1H), 3.44 (s, 3H), 2.42 (br s, 2H), 2.30 (s, 6H), 2.18 - 2.14 (m, 2H), 1.99 - 1.96 (m, 1H), 1.88 (dd, J = 14.6, 7.1 Hz, 2H), 1.67 (br s, 1H), 1.59 - 1.53 (m, 2H), 1.43 (br s, 2H), 1.28 - 1.26 (m, 5H), 0.83 - 0.89 (m, 6H).

[0317] Example 11 (B10): N 6 ,N6 -Diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [ka]

[0318] Step 1: Compound B6.2 (5.0 g) was dissolved in dichloromethane (100 ml), acetaldehyde (3.2 g) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to react. Sodium triacetoxyborohydride (12.8 g) was added to the reaction solution in batches, and after the addition was completed, the reaction system was stirred at room temperature for 1 hour to react. Completion of the reaction was confirmed by LCMS. Saturated aqueous ammonium chloride solution (20 ml) was added to the reaction solution, and the mixture was stirred for 1 hour, spin-dried, and filtered. The filtrate was separated and purified by reverse phase C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound B10.1 (4.57 g), which was a white solid.

[0319] LCMS (ESI) [M+H] + = 436.4;

[0320] 1 H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.38 - 7.26 (m, 5H), 5.08 - 4.99 (m, 2H), 4.00 (dd, J = 12.6, 6.5 Hz, 1H), 3.86 (dd, J = 8.6, 6.8 Hz, 1H), 2.74 (dd, J = 14.0, 6.9 Hz, 4H), 2.64 - 2.54 (m, 2H), 2.05 - 1.94 (m, 1H), 1.72 - 1.52 (m, 2H), 1.52 - 1.38 (m, 2H), 1.38 - 1.18 (m, 2H), 1.04 (t, J = 7.1 Hz, 6H), 0.87 - 0.81 (m, 6H).

[0321] Step 2: Compound B10.1 (1.6 g, 3.68 mmol) was dissolved in methanol (80 ml) at room temperature, and then Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred under hydrogen gas at room temperature for 12 hours. Completion of the reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B10.2 (900 mg, yield: 82%), which was an off-white solid.

[0322] 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (br s, 1H), 4.02 - 3.99 (m, 1H), 3.10 (d, J = 4.5 Hz, 1H), 2.65 (q, J = 7.1 Hz, 4H), 2.55 - 2.51 (m, 2H), 2.06 - 1.93 (m, 1H), 1.73 - 1.54 (m, 2H), 1.47 - 1.38 (m, 2H), 1.30 - 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

[0323] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 ml), and then HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added in sequence. The mixture was stirred at room temperature for 20 minutes, and then compound B10.2 (301 mg, 1 mmol) was added and the mixture was stirred at room temperature for 30 minutes. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound N 6 ,N 6 -Diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (B10, 280 mg, yield: 51%) was obtained as a white solid.

[0324] LCMS (ESI) [M+H] + = 552.3;

[0325] 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 4.18 (dd, J = 8.8, 6.8 Hz, 1H), 4.02 (dd, J = 12.8, 7.2 Hz, 1H), 3.41 (s, 3H), 2.74 - 2.69 (m, 4H), 2.62 - 2.52 (m, 4H), 2.44 - 2.29 (m, 2H), 2.04 - 1.94 (m, 1H), 1.86 - 1.77 (m, 2H), 1.72 - 1.54 (m, 2H), 1.51 - 1.39 (m, 2H), 1.33 - 1.23 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H), 0.87 - 0.82 (m, 6H).

[0326] Example 12 (B11): N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine [ka]

[0327] Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (500 mg, 1.475 mmol) was dissolved in N,N-dimethylformamide (10 ml), then HATU (560 mg, 1.475 mmol), N,N-diisopropylethylamine (476 mg, 3.688 mmol) were added, and the mixture was stirred for 30 minutes. Compound B10.2 (444 mg, 1.475 mmol) was then added, and the reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (B11, 330 mg, yield: 34%) was obtained as a white solid.

[0328] LCMS (ESI) [M+H] + = 623.4;

[0329] 1H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 9.04 (s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 7.1 Hz, 1H), 4.47 (t, J = 7.0 Hz, 2H), 4.14 (dd, J = 8.8, 6.6 Hz, 1H), 4.06 - 3.95 (m, 1H), 3.44 (s, 3H), 2.67 - 2.64 (m, 4H), 2.55 (t, J = 7.4 Hz, 2H), 2.21 - 2.10 (m, 2H), 2.02 - 1.94 (m, 1H), 1.92 - 1.84 (m, 2H), 1.72 - 1.63 (m, 1H), 1.72 - 1.63 (m, 3H), 1.59 - 1.54 (m, 2H), 1.32 - 1.21 (m, 4H), 1.01 (t, J = 7.1 Hz, 6H), 0.81 (dd, J = 9.6, 6.8 Hz, 6H).

[0330] Example 13 (B12): N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [ka]

[0331] Step 1: Compound B6.2 (5.0 g) was dissolved in dichloromethane (100 ml), n-propionaldehyde (4.2 g) was added to the reaction solution, and the reaction was stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g) was added to the reaction solution in batches, and the reaction system was stirred at room temperature for 1 hour. The completion of the reaction was confirmed by LCMS. Saturated aqueous ammonium chloride solution (20 ml) was added to the reaction solution, and the reaction was stirred for 1 hour, spin-dried, and filtered. The filtrate was separated and purified using a reversed-phase C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound B12.1 (4.57 g, yield: 82.0%), which was a white solid.

[0332] LCMS (ESI) [M+H] + = 464.0;

[0333] 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 7.3 Hz, 1H), 7.38 - 7.28 (m, 5H), 5.04 (d, J = 1.7 Hz, 2H), 4.12 - 4.02 (m, 1H), 3.94 - 3.82 (m, 1H), 2.65 - 2.52 (m, 6H), 2.06 - 1.94 (m, 1H), 1.76 - 1.64 (m, 1H), 1.64 - 1.53 (m, 1H), 1.52 - 1.40 (m, 6H), 1.34 - 1.18 (m, 2H), 0.92 - 0.80 (m, 12H).

[0334] Step 2: Compound B12.1 (2.0 g, 4.32 mmol) was dissolved in methanol (80 ml) at room temperature, and then Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred under hydrogen gas at room temperature for 12 hours. Completion of the reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B12.2 (1.2 g, yield: 85.5%), which was a white solid.

[0335] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 ml), then HATU (142 mg, 0.373 mmol), N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added, and the reaction system was stirred for 30 minutes, then compound B12.2 (122 mg, 0.371 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (50 mg, yield: 28%) was obtained as a pale yellow solid.

[0336] LCMS (ESI) [M+H] + = 580.0;

[0337] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 - 7.93 (m, 2H), 4.24 - 4.16 (m, 1H), 4.10 (d, J = 5.2 Hz, 1H), 3.41 (s, 3H), 2.79 - 2.64 (m, 6H), 2.55 (t, J = 7.1 Hz, 2H), 2.45 - 2.26 (m, 2H), 2.06 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.76 - 1.66 (m, 1H), 1.64 - 1.57 (m, 1H), 1.57 - 1.42 (m, 6H), 1.37 - 1.24 (m, 2H), 0.93 - 0.78 (m, 12H).

[0338] Example 14 (B13): N 6 ,N 6 -Dipropyl-N 2-((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine [ka]

[0339] Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (500 mg, 1.475 mmol) was dissolved in N,N-dimethylformamide (10 ml), then HATU (560 mg, 1.475 mmol), N,N-diisopropylethylamine (476 mg, 3.688 mmol) were added, the reaction system was stirred for 30 minutes, then compound B12.2 (485 mg, 1.475 mmol) was added, the reaction solution was stirred at room temperature for 1 hour, and after the completion of the reaction was detected by LCMS, the reaction solution was directly purified by reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (B13, 320 mg, yield: 33%) was obtained as a white solid.

[0340] LCMS (ESI) [M+H] + = 651.5;

[0341] 1H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 9.00 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 4.47 (t, J = 6.9 Hz, 2H), 4.21 - 4.11 (m, 1H), 4.09 - 4.01 (m, 1H), 3.44 (s, 3H), 2.45 - 2.35 (m, 6H), 2.23 - 2.09 (m, 2H), 1.97 - 1.86 (m, 3H), 1.72 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.43 - 1.34 (m, 6H), 1.32 - 1.22 (m, 4H), 0.86 - 0.77 (m, 12H).

[0342] Example 15 (B14): tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hexa-5-ynamido)-6-oxohexyl)carbamate [ka]

[0343] Step 1: Compound 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoic acid (3.70 g, 15.7 mmol), diisopropylethylamine (11.0 mL, 62.8 mmol) and HBTU (8.90 g, 23.6 mmol) were added in sequence to N,N-dimethylformamide (30 ml), and the reaction system was reacted at room temperature for 30 minutes. Then, compound Boc-protected lysine (3.86 g, 15.7 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. The completion of the reaction was detected by LCMS. The reaction solution was adjusted to PH=5 by adding aqueous citric acid solution (30 ml), extracted with dichloromethane (50 ml x 3), the organic phase was combined, dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain the target compound B14.1 (7.0 g), which was a yellow oil. LCMS (ESI) [M+H] + = 465.1.

[0344] Step 2: Compound B14.1 (7.00 g, 5.60 mmol), diisopropylethylamine (10.7 mL, 60.4 mmol) and HBTU (8.60 g, 22.7 mmol) were added in sequence to N,N-dimethylformamide (100 ml), followed by p-aminobenzyl alcohol (3.71 g, 30.2 mmol), and the reaction was allowed to react at room temperature for 2 hours. Completion of the reaction was detected by LCMS. Ethyl acetate (300 ml) was added to the reaction solution, washed with saturated saline (100 ml × 3), dried over anhydrous sodium sulfate, filtered by suction, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamido)-6-oxohexyl)carbamate (B14, 6.00 g, yield: 69.9%), which was a yellow oil.

[0345] LCMS (ESI) [M+H] + = 570.1;

[0346] 1 H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.46 (s, 2H), 7.51 - 7.47 (m, 2H), 7.31 - 7.23 (m, 3H), 7.02 - 6.95 (m, 1H), 4.83 (br s, 1H), 4.63 - 4.60 (m, 2H), 3.19 - 2.96 (m, 4H), 2.57 (s, 3H), 2.52 - 2.48 (m, 2H), 2.47 - 2.41 (m, 2H), 1.99 - 1.92 (m, 2H), 1.87 - 1.77 (m, 2H), 1.76 - 1.63 (m, 2H), 1.43 (s, 9H).

[0347] Example 16 (B15): tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate [ka]

[0348] Step 1: Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (1.60 g, 5.21 mmol), diisopropylethylamine (2.68 mL, 20.8 mmol) and HBTU (2.97 g, 7.82 mmol) were added in sequence to N,N-dimethylformamide (30 ml), and the reaction system was reacted at room temperature for 30 minutes. Then, Boc-protected lysine (1.28 g, 5.21 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. The completion of the reaction was detected by LCMS. The reaction solution was adjusted to PH=5 by adding aqueous citric acid solution (30 ml), extracted with dichloromethane (50 ml x 3), the organic phase was combined, dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain target compound B15.1 (3.0 g, crude product), which was a yellow oil.

[0349] LCMS (ESI) [M+H] + = 536.0;

[0350] Step 2: Compound B15.1 (3.00 g, 5.60 mmol), diisopropylethylamine (2.89 g, 22.4 mmol) and HBTU (3.19 g, 8.40 mmol) were added in sequence to N,N-dimethylformamide (30 ml). Then, p-aminobenzyl alcohol (1.38 g, 11.2 mmol) was added and the reaction was allowed to react at room temperature for 2 hours. The completion of the reaction was detected by LCMS. Ethyl acetate (200 ml) was added to the reaction solution, washed with saturated saline (80 ml × 3), dried over anhydrous sodium sulfate, filtered by suction, the filtrate was concentrated under reduced pressure to a solid, and the crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate (B15, 2.90 g), which was a yellow oil.

[0351] LCMS (ESI) [M+H] + = 641.1;

[0352] 1 H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.95 (s, 2H), 7.94 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 6.83 - 6.77 (m, 1H), 4.93 - 4.80 (m, 1H), 4.64 - 4.58 (m, 2H), 4.40 - 4.29 (m, 2H), 3.12 - 2.97 (m, 4H), 2.60 (s, 3H), 2.31 - 2.23 (m, 2H), 1.96 - 1.83 (m, 4H), 1.76 - 1.61 (m, 4H), 1.41 (s, 9H), 1.37 - 1.29 (m, 4H).

[0353] Example 17 (B16): N 6 ,N 6 -Dibutyl-N 2-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [ka]

[0354] Step 1: Compound B6.2 (10.0 g) was dissolved in dichloromethane (200 ml) solution, n-butylaldehyde (10.4 g) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to react. Sodium triacetoxyborohydride (25.6 g) was added to the reaction solution in batches, and the reaction system was stirred at room temperature for 1 hour to react. Completion of the reaction was confirmed by LCMS. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was stirred for 1 hour, spin-dried, and filtered. The filtrate was separated and purified using a reverse phase C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound B16.1 (4.9 g), which was a white solid.

[0355] LCMS (ESI) [M+H] + = 492.7;

[0356] 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.30 (m, 5H), 5.14 - 5.08 (m, 2H), 4.33 - 4.30 (m, 1H), 4.14 - 4.12 (m, 1H), 2.94 - 2.80 (m, 6H), 2.13 - 2.11 (m, 1H), 1.85 - 1.82 (m, 2H), 1.58 - 1.55 (m, 4H), 1.40 - 1.22 (m, 8H), 0.98 - 0.87 (m, 12H).

[0357] Step 2: Compound B16.1 (5.0 g) was dissolved in methanol (100 ml) at room temperature, then Pd / C (10%, 1 g) was added to the reaction solution, and the reaction was stirred under hydrogen gas for 12 hours. The completion of the reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B16.2 (3.68 g), which was a white solid.

[0358] LCMS (ESI) [M+H] + = 358.3.

[0359] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1.0 g) was dissolved in N,N-dimethylformamide (15 ml), then added successively to N,N-diisopropylethylamine (1.2 g) and HATU (1.4 g), the reaction system was stirred for 30 minutes, and then compound B16.2 (1.3 g) was added, the reaction solution was stirred at room temperature for 1 hour, and after the completion of the reaction was detected by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% aqueous trifluoroacetic acid, acetonitrile) to obtain the target compound N 6 ,N 6 -Dibutyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (B16, 500 mg) was obtained as a yellow solid.

[0360] LCMS (ESI) [M+H] + = 608.7;

[0361] 1H NMR (400 MHz, DMSO) δ 9.37 (s, 1H), 9.13 (s, 2H), 8.19 (d, J = 7.3 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 4.25 - 4.20 (m, 1H), 4.20 - 4.13 (m, 1H), 3.42 (s, 3H), 3.06 - 2.99 (m, 6H), 2.57 - 2.53 (m, 2H), 2.41 - 2.30 (m, 2H), 1.99 - 1.95 (m, 2H), 1.87 - 1.79 (m, 2H), 1.78 - 1.71 (m, 1H), 1.62 - 1.56 (m, 4H), 1.34 - 1.32 (m, 8H), 0.94 - 0.85 (m, 12H).

[0362] Example 18 (B17): (S)-1-Ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamido)butanamido)piperidine-4-carboxylic acid [ka]

[0363] Step 1: Compound B17.1 (5.0 g, 20.49 mmol) was dissolved in DMF (100 ml), and N-((benzyloxy)carbonyl)-L-valinic acid 2,5-dioxopyrrolidine-N-hydroxyester (7.13 g, 20.49 mmol) was added to the reaction solution, and the mixture was stirred at 100°C for 48 hours to react. Completion of the reaction was confirmed by LCMS. The reaction solution was separated and purified using a reversed-phase C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5% to 55%) to obtain the target compound B17.2 (1.3 g, yield: 13.3%), which was a yellow solid.

[0364] LCMS (ESI) [M-boc] + = 378.2;

[0365] 1H NMR (400 MHz, DMSO) δ 7.38 - 7.33 (m, 5H), 7.32 - 7.27 (m, 1H), 7.20 (d, J = 9.0 Hz, 1H), 5.14 - 5.02 (m, 2H), 3.97 - 3.87 (m, 1H), 3.70 - 3.59 (m, 2H), 3.08 - 2.95 (m, 2H), 2.04 - 1.97 (m, 1H), 1.95 - 1.87 (m, 2H), 1.76 - 1.69 (m, 2H), 1.40 (s, 9H), 0.91 - 0.84 (m, 6H).

[0366] Step 2: Compound B17.2 (1.3 g, 2.71 mmol) was dissolved in ethyl acetate (10 ml) at room temperature, and then dioxane-HCl (3N, 10 ml) was added to the reaction solution, and the reaction was stirred at room temperature for 1 hour. The completion of the reaction was confirmed by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound B17.3 (1.06 g, yield: 94.4%), which was a yellow solid.

[0367] LCMS (ESI) [M+H] + = 378.4.

[0368] Step 3: Compound B17.3 (1.06 g, 2.81 mmol) was dissolved in dichloromethane (10 ml), acetaldehyde (0.75 g, 16.87 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to react. Sodium triacetoxyborohydride (3 g, 14.05 mmol) was added to the reaction solution in batches, and the reaction system was stirred at room temperature for 1 hour to react. The reaction was confirmed to be complete by LCMS. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was stirred for 1 hour. The mixture was evaporated under reduced pressure to remove dichloromethane, and the remaining aqueous phase was separated by reverse phase (acetonitrile / 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound B17.4 (1.13 g, yield: 99.23%), which was a white solid.

[0369] LCMS (ESI) [M+H] += 406.1;

[0370] 1H NMR (400 MHz, MeOD) δ 7.42 - 7.25 (m, 5H), 5.22 - 5.06 (m, 2H), 3.91 (d, J = 7.0 Hz, 1H), 3.52 - 3.36 (m, 2H), 3.25 - 2.93 (m, 4H), 2.66 - 2.54 (m, 1H), 2.47 - 2.36 (m, 1H), 2.27 - 2.14 (m, 2H), 2.13 - 2.06 (m, 1H), 1.35 - 1.29 (m, 4H), 0.98 - 0.90 (m, 6H).

[0371] Step 4: Compound B17.4 (1.13 g, 2.78 mmol) was dissolved in methanol (20 ml) solution at room temperature, then Pd / C (0.5 g) was added to the reaction solution, and the reaction was stirred under hydrogen gas for 12 hours. The completion of the reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound B17.5 (0.58 g, yield: 76.18%), which was a white solid. LCMS (ESI) [M+H] + = 272.4.

[0372] Step 5: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1 g, 3.73 mmol) was dissolved in N,N-dimethylformamide (5 ml), then added successively to N,N-diisopropylethylamine (1.2 g, 9.32 mmol), HATU (1.42 g, 3.73 mmol), and the reaction solution was stirred for 30 minutes, and then compound B17.5 (1.01 g, 3.73 mmol) was added, and the reaction solution was stirred at room temperature. After stirring for 1 hour and detecting the completion of the reaction by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% aqueous trifluoroacetic acid, acetonitrile) to obtain the target compound (S)-1-ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)piperidine-4-carboxylic acid (B17, 500 mg, yield: 28.7%), which was a yellow solid.

[0373] LCMS (ESI) [M+H] + = 522.5;

[0374] 1 H NMR (400 MHz, DMSO) δ 9.12 (s, 2H), 8.08 (s, 1H), 7.88 (d, J = 9.0 Hz, 1H), 4.28 - 4.25 (m, 1H), 3.41 (s, 3H), 2.89 - 2.78 (m, 2H), 2.57 - 2.52 (m, 4H), 2.45 - 2.30 (m, 4H), 2.07 - 2.01 (m, 2H), 1.99 - 1.96 (m, 1H), 1.95 - 1.88 (m, 2H), 1.85 - 1.79 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.88 - 0.83 (m, 6H).

[0375] Example 19 (B18): 4-((S)-2-((S)-2-(2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamido)-3-methylbutanamido)-6-(dimethylamino)hexanamido)benzyl(4-nitrophenyl)carbonate [ka]

[0376] Step 1: Compound B18.1 (500 mg, 4.39 mmol) was dissolved in ultra-dry dichloromethane (5 mL), and boron tribromide (1.15 g, 4.6 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was warmed to room temperature and stirred for 24 hours. The mixture was cooled to 0°C, and methanol (5 ml) was added dropwise to the reaction solution to quench the reaction. The reaction solution was stirred at room temperature for 24 hours. Saturated aqueous sodium bicarbonate solution (30 ml) was added to the reaction solution, which was then extracted with dichloromethane (30 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off to obtain B18.2 (830 mg, yield: 92.5%) as a brown oily liquid.

[0377] 1 H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.40 - 3.27 (m, 2H), 2.20 - 2.09 (m, 2H), 1.21 (s, 6H).

[0378] Step 2: Compound 18.2 (650 mg, 3.13 mmol) was dissolved in DMF (10 mL), sodium azide (1.02 g, 15.63 mmol) was added, the mixture was heated to 80° C. and stirred overnight, then cooled to room temperature, water (30 ml) was added to the reaction solution, and then extracted with MTBE (30 ml×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain target compound B18.3 (470 mg, yield: 88.9%), which was a pale yellow oily liquid.

[0379] 1 H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.31 - 3.23 (m, 2H), 1.89 - 1.81 (m, 2H), 1.22 (s, 6H).

[0380] Step 3: Compound 18.3 (365 mg, 2.13 mmol), 5-ethynyl-2-(methylthio)pyrimidine (320 mg, 2.13 mmol) were dissolved in tert-butanol (5 mL) and water (5 mL), and vitamin C sodium (84 mg, 0.43 mmol), copper sulfate anhydride (34 mg, 0.21 mmol) were added and stirred at room temperature for 1 hour. Water (25 ml) was added to the reaction solution, and then extracted with ethyl acetate (30 ml x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, spin-dried, and the residue was purified by silica gel column chromatography (PE:EA = 3:1) to obtain the target compound B18.4 (600 mg, yield: 88.2%), which was a white solid.

[0381] LCMS (ESI) [M+H] + = 322.1

[0382] 1 H NMR (400 MHz, CDCl3) δ 8.95 (s, 2H), 7.85 (s, 1H), 4.53 - 4.39 (m, 2H), 3.69 (s, 3H), 2.61 (s, 3H), 2.29 - 2.17 (m, 2H), 1.31 (s, 6H).

[0383] Step 4: Compound 18.4 (500 mg, 1.60 mmol) was dissolved in tetrahydrofuran (5 mL) and water (5 mL), lithium hydroxide (112 mg, 4.70 mmol) was added, and the mixture was stirred at room temperature for 5 hours. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 3-4, and a large amount of white solid precipitated. The cake was filtered to obtain the target compound B18.5 (450 mg, yield: 93.5%), which was a white solid.

[0384] LCMS (ESI) [M+H] + = 308.0

[0385] 1 H NMR (400 MHz, DMSO) δ 12.43 (s, 1H), 9.06 (s, 2H), 8.76 (s, 1H), 4.55 - 4.31 (m, 2H), 2.56 (s, 3H), 2.15 - 2.04 (m, 2H), 1.20 (s, 6H).

[0386] Step 5: Compound B18.5 (1.5 g, 4.90 mmol) was dissolved in tetrahydrofuran (10 mL) and water (10 mL), and Oxane (15.0 g, 24.5 mmol) was added and stirred at room temperature overnight. The reaction solution was poured into water (200 ml), filtered, and the cake was the target compound B18.6 (1.6 g, yield: 93.5%), which was a white solid.

[0387] LCMS (ESI) [M+H] + = 340.1

[0388] 1 H NMR (400 MHz, DMSO) δ 9.48 (s, 2H), 8.98 (s, 1H), 4.54 - 4.48 (m, 2H), 3.44 (s, 3H), 2.16 - 2.09 (m, 2H), 1.21 (s, 6H).

[0389] Step 6: Compound B18.6 (1.9 g, 5.32 mmol) was dissolved in DMF (40 ml), and HATU (2.13 g, 5.32 mmol) and DIEA (1.7 g, 13.3 mmol) were added. The reaction solution was stirred at room temperature for 30 minutes and then cooled to 5° C. with N 2 -(L-valyl)-N 6 ,N 6-Dimethylamino-L-lysine (1.45 g, 5.32 mmol) was added to the reaction solution and stirred for 1 h. The reaction solution was directly purified by reverse phase chromatography (HO:ACN=5%~100%) to obtain the target compound B18.7 (1.5 g, yield: 44.5%) as a white solid.

[0390] LCMS (ESI) [M+H] + = 595.5

[0391] 1 H NMR (400 MHz, DMSO) δ 9.51 (s, 2H), 9.05 (s, 1H), 7.89 (d, J = 7.1 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 4.45 - 4.35 (m, 2H), 4.13 (t, 1H), 4.07-4.09 (m, 1H), 3.44 (s, 3H), 2.34 (t, J = 7.3 Hz, 2H), 2.23 (s, 6H), 2.19 - 2.06 (m, 4H), 1.73 - 1.57 (m, 2H), 1.45 - 1.36 (m, 2H), 1.31 - 1.27 (m, 1H), 1.22 (d, J = 9.6 Hz, 6H), 0.91 - 0.86 (m, 6H).

[0392] Step 7: Compound 18.7 (360 mg, 0.61 mmol), (4-aminophenyl)ethanol (75 mg, 0.61 mmol) were dissolved in DMF (5 ml), HATU (230 mg, 0.61 mmol) and DIEA (195 mg, 1.52 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase chromatography (HO:ACN=5%~100%) to obtain the target compound B18.8 (255 mg, yield: 60.1%), which was a white solid.

[0393] LCMS (ESI) [M+H] + = 700.4

[0394] 1H NMR (400 MHz, DMSO) δ 9.93 (d, J = 87.4 Hz, 1H), 9.47 (s, 1H), 9.39 (s, 1H), 8.89 (d, J = 57.3 Hz, 1H), 8.54 - 8.16 (m, 1H), 7.68 (d, J = 7.8 Hz, 0.5H), 7.56 - 7.48 (m, 2H), 7.47 (d, J = 8.3 Hz, 0.5H), 7.19 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 5.10 - 5.04 (m, 1H), 4.44 - 4.36 (m, 4H), 4.21 - 4.09 (m, 1H), 3.45 (s, 3H), 2.75 - 2.65 (m, 2H), 2.23 - 2.04 (m, 4H), 1.83 - 1.63 (m, 2H), 1.59 - 1.51 (m, 2H), 1.42 - 1.35 (m, 1H), 1.35 - 1.16 (m, 12H), 0.94 - 0.87 (m, 6H).

[0395] Step 8: Compound 18.8 (250 mg, 0.36 mmol) and 4-nitrophenyl carbonochloridate (288 mg, 1.43 mmol) were dissolved in DCM (10 mL), TEA (72 mg, 0.72 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated, and the residue was purified by reverse phase chromatography (HO:ACN=5%-100%) to give B18 (120 mg, yield: 38.9%) as a yellow solid.

[0396] LCMS (ESI) [M+H] + = 865.2;

[0397] 1H NMR (400 MHz, DMSO) δ 10.04 (d, J = 73.3 Hz, 1H), 9.46 (s, 1H), 9.36 (s, 1H), 8.86 (d, J = 56.6 Hz, 1H), 8.31 (d, J = 9.1 Hz, 2H), 8.08 (d, J = 9.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.59 - 7.55 (m, 3H), 7.42 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 9.1 Hz, 1H), 5.19 (d, J = 14.8 Hz, 2H), 4.45 - 4.38 (m, 2H), 4.37 - 4.28 (m, 1H), 4.21 - 4.14 (m, 1H), 3.44 (s, 3H), 2.18 - 2.12 (m, 3H), 2.10 - 2.06 (m, 8H), 1.81 - 1.63 (m, 2H), 1.44 - 1.32 (m, 4H), 1.23 (d, J = 7.4 Hz, 7H), 0.94 - 0.88 (m, 6H).

[0398] Example 20 (B19): 4-((S)-2-((S)-2-(2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate [ka]

[0399] Step 1: Compound 19.1 (447 mg, 1.39 mmol) and 18.6 (500 mg, 1.39 mmol) were dissolved in DMF (5 ml), HATU (500 mg, 1.39 mmol) and DIEA (425 mg, 3.30 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase chromatography (HO:ACN=5%~100%) to obtain the target compound B19.2 (900 mg, yield: 97.0%) as a white solid.

[0400] LCMS (ESI) [M+H] + = 701.5

[0401] 1 H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 9.46 (s, 2H), 8.96 (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.53 - 7.50 (m, 2H), 7.47 (d, J = 8.6 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H), 6.05 (t, J = 5.7 Hz, 1H), 5.43 (s, 2H), 5.08 (t, J = 5.7 Hz, 1H), 4.45 - 4.38 (m, 5H), 4.18 (t, J = 8.2 Hz, 1H), 3.63 - 3.58 (m, 1H), 3.44 (s, 3H), 3.07 - 2.94 (m, 2H), 2.23 - 2.01 (m, 4H), 1.80 - 1.71 (m, 2H), 1.24 (d, J = 9.5 Hz, 6H), 0.92 - 0.87 (m, 6H).

[0402] Step 2: Compound B19.2 (400 mg, 0.57 mmol) and 4-nitrophenyl carbonochloridate (574 mg, 2.86 mmol) were dissolved in DCM (10 mL), TEA (173 mg, 1.71 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated, and the residue was purified by reverse phase chromatography (HO:ACN=5%-100%) to obtain the target compound B19 (150 mg, yield: 30.4%) as a yellow solid.

[0403] LCMS (ESI) [M+H] + = 866.5

[0404] 1 H NMR (400 MHz, DMSO) δ 10.18 (s, 1H), 9.47 (s, 2H), 8.95 (s, 1H), 8.32 (d, J = 9.2 Hz, 1H), 8.18 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.63 - 7.56 (m, 3H), 7.55 - 7.47 (m, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H), 6.04 (t, J = 4.4 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 1H), 4.48 - 4.37 (m, 4H), 4.22 - 4.15 (m, 1H), 3.44 (s, 3H), 3.05 - 2.96 (m, 2H), 2.20 - 2.09 (m, 3H), 2.04 - 1.98 (m, 2H), 1.51 - 1.43 (m, 2H), 1.25 (d, 6H), 0.92 - 0.87 (m, 6H).

[0405] II. Synthesis of drug-linker compounds Example 21 (2.1): 4-((S)-2-((S)-2-(2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(elibrinyl)carbamate (DL-001) [ka]

[0406] Compound B18 (36 mg, 0.041 mmol) and compound Eribulin (20 mg, 0.027 mmol) were dissolved in DMF (2 ml). DIEA (7 mg, 0.054 mmol) was then added, and after the addition was completed, the reaction solution was stirred at room temperature for 1 hour. The crude product was purified by preparative chromatography (0.01% FA in water, MeCN) to obtain the target compound DL001 (21.8 mg, yield: 52.6%), which was a white solid.

[0407] LCMS (ESI) [M+H] + = 1455.6;

[0408] 1H NMR (400 MHz, DMSO) δ 10.01 (d, J = 69.6 Hz, 1H), 9.46 (s, 1H), 9.39 (s, 1H), 8.88 (d, J = 47.0 Hz, 1H), 8.50 (d, J = 7.3 Hz, 0.5H), 8.37 (s, 2H), 8.16 (d, J = 6.7 Hz, 0.5H), 7.67 - 7.46 (m, 3H), 7.23 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 5.4 Hz, 1H), 5.05 (s, 1H), 4.99 (s, 1H), 4.90 (d, J = 3.6 Hz, 1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.55 (t, J = 4.0 Hz, 1H), 4.43 - 4.33 (m, 3H), 4.26 (d, J = 10.3 Hz, 1H), 4.21 - 4.14 (m, 2H), 4.11 (d, J = 10.6 Hz, 4H), 4.01 (d, J = 9.8 Hz, 1H), 3.84 - 3.73 (m, 4H), 3.72 - 3.65 (m, 3H), 3.44 (s, 3H), 3.27 - 3.22 (m, 5H), 2.96 (t, J = 5.6 Hz, 2H), 2.84 (d, J = 9.5 Hz, 1H), 2.80 - 2.64 (m, 3H), 2.60 - 2.55 (m, 1H), 2.36 - 2.29 (m, 2H), 2.27 - 2.23 (m, 2H), 2.21 - 2.11 (m, 6H), 2.08 (d, J = 4.8 Hz, 6H), 2.06 - 2.03 (m, 1H), 2.03 - 1.95 (m, 2H), 1.94 - 1.88 (m, 4H), 1.74 - 1.60 (m, 7H), 1.56 - 1.45 (m, 3H), 1.38 - 1.28 (m, 6H), 1.24 - 1.20 (m, 6H), 1.03 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 12.0 Hz, 1H), 0.92 - 0.86 (m, 6H).

[0409] Example 22 (2.2): 4-((S)-2-((S)-2-(2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(elibrinyl)carbamate (DL-002) [ka]

[0410] Compound B19 (48 mg, 0.055 mmol) and compound eribulin (20 mg, 0.027 mmol) were dissolved in DMF (2 ml), then DIEA (8.7 mg, 0.068 mmol) was added, and after the addition was completed, the reaction solution was stirred at room temperature for 1 hour. The crude product was purified by preparative chromatography (0.01% FA in water, MeCN) to obtain the target compound DL-002 (13.34 mg, yield: 33.6%), which was a white solid.

[0411] LCMS (ESI) [M+H] + = 1456.6;

[0412] 1H NMR (400 MHz, DMSO) δ 10.07 (s, 1H), 9.46 (s, 2H), 8.94 (s, 1H), 8.37 (s, 1H), 8.14 (d, J = 7.2 Hz, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.07 (t, J = 5.0 Hz, 1H), 6.00 (t, J = 5.3 Hz, 1H), 5.42 (s, 2H), 5.05 (s, 1H), 5.00 (s, 1H), 4.95 - 4.87 (m, 2H), 4.84 (s, 1H), 4.76 (s, 1H), 4.64 (s, 1H), 4.58 - 4.54 (m, 1H), 4.47 - 4.38 (m, 3H), 4.26 (d, J = 10.4 Hz, 1H), 4.21 - 4.14 (m, 2H), 4.11 (s, 3H), 4.05 - 3.98 (m, 1H), 3.84 - 3.73 (m, 2H), 3.72 - 3.65 (m, 1H), 3.57 - 3.46 (m, 4H), 3.44 (s, 3H), 3.25 (s, 4H), 3.06 - 2.91 (m, 4H), 2.84 (d, J = 9.6 Hz, 1H), 2.81 - 2.67 (m, 2H), 2.61 - 2.54 (m, 1H), 2.38 - 2.04 (m, 10H), 2.02 - 1.87 (m, 6H), 1.75 - 1.61 (m, 6H), 1.56 - 1.43 (m, 4H), 1.39 - 1.27 (m, 4H), 1.23 (d, J = 7.1 Hz, 6H), 1.18 (d, J = 14.7 Hz, 1H), 1.08 - 1.01 (m, 3H), 0.98 (d, J = 12.1 Hz, 1H), 0.93 - 0.86 (m, 6H).

[0413] 3. Antibody production and identification Example 23 (3.1.1) Production of B7H3 antigen and anti-B7H3 antibody The nucleic acid sequence of amino acids 29 to 245 of human 2Ig B7H3 (NCBI protein number: NP_001316557.1) was selected, and a 6×His detection tag was added to the C-terminus of the nucleic acid sequence, which was named human B7H3-2IgG-his. The nucleic acid sequence of amino acids 27 to 461 of human 4Ig B7H3 (Uniprot protein number: Q5ZPR3-1) was selected, and a 6×His purification tag was added to the C-terminus of the nucleic acid sequence, which was named human B7H3-4IgG-his. The nucleic acid sequence of amino acids 27 to 465 of monkey B7H3 4Ig (Uniprot protein number: F7G5V3) was selected, and a 6×His purification tag was added to the C-terminus of the nucleic acid sequence, which was named monkey B7H3-4IgG-his. B7H3-C1 refers to the antibody with the number M30-H1-L4 in CN103687945B, and B7H3-C2 refers to the antibody with the number mAb-C-DUBA in CN109069633A.

[0414] The genes of the three B7H3 antigens were synthesized, and the genes of the B7H3-C1 and B7H3-C2 antibodies (Sangon Biotech (Shanghai) Co., Ltd.) were synthesized by codon optimization and constructed into a PTT5 expression vector, and a number of plasmids were extracted. The extracted and prepared expression vectors were transiently introduced and expressed in HEK293F for 7 days, and the expression supernatant was prepared using Protein A to obtain the antigen protein and the B7H3-C1 and B7H3-C2 antibodies.

[0415] The amino acid sequence of IgG1 constant region (SEQ ID NO:20) was added to the VH amino acid sequence of 1D1 and 2E3, respectively, by codon optimization and gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and then constructed into PTT5 vector, which were named PTT5-01-CH and PPT5-02-CH, respectively. The amino acid sequence of Kappa constant region (SEQ ID NO:21) was added to the VL amino acid sequence of 1D1 and 2E3, respectively, by codon optimization and gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and then constructed into PTT5 vector, which were named PTT5-01-CL and PTT5-01-CL, respectively. Antibody B7H3 antibody expression plasmid PTT5-01-CH / PTT5-01-CL, PTT5-02-CH / PTT5-02-CL were co-transfected into HEK293F cells using PEI max reagent, and expressed for 7 days in a shaker at 37°C with 5% CO2. The supernatant was collected and purified by ProA magnetic beads to obtain anti-B7H3 antibodies, which were named 1D1-01 (whose heavy chain sequence is SEQ ID NO:3, its light chain sequence is SEQ ID NO:4) and 2E3-02 (whose heavy chain sequence is SEQ ID NO:7, its light chain sequence is SEQ ID NO:8), respectively.

[0416] Example 24 (3.1.2) Protein affinity detection of anti-B7H3 antibody The affinity of B7H3 fully human antibody to human B7H3-4IgG-his and monkey B7H3-4IgG-his proteins was detected by ELISA experimental method. Specific experimental steps: Human B7H3-4IgG-his or monkey B7H3-4IgG-his proteins were diluted to a final concentration of 1 μg / mL in carbonate buffer with a pH value of 9.6, and added to a 96-well ELISA plate at 100 μL / well, respectively, and coated at 4°C overnight. The coating solution was discarded, washed once with PBST, and 100 μL of PBS (containing 2% BSA) was added to each well, and blocked at 37°C for 1 hour. Then, B7H3 fully human antibody (starting from 10 μg, diluted 3-fold, two replicate wells) diluted with 100 μL of PBS (containing 2% BSA) was added to each well, and incubated at 37°C for 2 hours. After washing three times with PBST, horseradish peroxidase-labeled anti-human Fc antibody (diluted 1:10000 with PBS) was added at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed five times with PBST. TMB color development solution was added at 100 μL / well and color development was allowed to continue for 7 minutes at 37°C. The reaction was terminated by adding stop solution at 50 μL / well, and the absorbance at 450 nm was measured. EC 50 values ​​were calculated.

[0417] The results are shown in Figures 1A, 1B, 2A and 2B, and the detailed results are shown in Tables 1-1 and 1-2. 1D1-01, 2E3-02 and human B7H3-4 IgG all had high affinity, 43.56 pM and 17.54 pM, respectively, where 2E3-02 had a stronger affinity than B7H3-C1 at 51.85 pM, and 1D1-01, 2E3-02 and monkey B7H3-4 IgG had high binding ability, 35.82 pM and 27.72 pM, respectively.

[0418] Table 1-1. Protein affinity test results of 1D1-01 antibody [Table 9]

[0419] Table 1-2. Protein affinity test results of 2E3-02 antibody [Table 10]

[0420] Example 25 (3.1.4) Cell affinity test of anti-B7H3 antibody B7H3 protein is highly expressed in various solid tumor cells, and the affinity of the B7H3 fully human antibody to tumor cells was detected by FACS experimental method. Specific experimental steps: MCF-7 breast cancer cells, A549 human non-small cell lung cancer cells, and PC3 human prostate cancer cells grown to logarithmic growth phase were digested with trypsin and diluted with 5×10^ in PBS buffer containing 2% BSA. 5 The cells were resuspended at 100μL / mL and 100uL of the cell suspension was added to a 96-well plate. B7H3-C1, B7H3-C2, 1D1-01 and 2E3-02 were diluted 3-fold with PBS buffer containing 2% BSA at a starting concentration of 40μg / mL, then 100μL of the antibody suspension was added to the 96-well plate and mixed evenly. The cells were incubated at 4℃ for 1 hour, washed twice with 300μL / well of pre-chilled PBS, and centrifuged at 500g for 5 minutes. Fluorescent secondary antibodies APC anti-human IgG or PE anti-human IgG (purchased from Biolegend) at 1:50 were added at 100μL / well and mixed evenly. The cells were incubated at 4℃ for 0.5 hour, washed twice with 300μL / well of pre-chilled PBS, centrifuged at 500g for 5 minutes, resuspended in 500μl of PBS, and the mean fluorescent signal values ​​were detected using a Beckman flow cytometer. EC from the mean fluorescence signal 50 The values ​​were calculated, and the results are shown in Figures 3A, 3B, 4A, 4B and Figure 5, and specific data are shown in Tables 2-1 and 2-2.

[0421] Table 2-1. Cell affinity test results of 1D1-01 antibody [Table 11] Table 2-2. Cell affinity test results of 2E3-02 antibody [Table 12]

[0422] As shown in Tables 2-1 and 2-2, for MCF-7 tumor cells expressing B7H3 highly, the affinities of 1D1-01 and 2E3-02 were 0.524nM and 0.398nM, respectively, both of which were higher than B7H3-C1, and the maximum fluorescence signal values ​​were 12% and 23% higher than B7H3-C1, where the maximum fluorescence signal value of 2E3-02 was 2.78 times higher than B7H3-C2. For A549 tumor cells expressing B7H3 moderately, the affinities of 1D1-01 and 2E3-02 were 0.322nM and 0.150nM, respectively, both of which were higher than B7H3-C1, and the maximum fluorescence signal values ​​were 36% and 77% higher than B7H3-C1, respectively. For PC-3 tumor cells with low B7H3 expression, the affinity of 2E3-02 (0.06 nM) was much higher than that of B7H3-C1 (0.618 nM) and higher than that of B7H3-C2 (0.167 nM), while the maximum fluorescence signal values ​​were 1.51 and 4.48 times higher than those of B7H3-C1 and B7H3-C2, respectively. In conclusion, the anti-B7H3 antibodies 1D1-01 and 2E3-02 have better affinity to tumor cells than B7H3-C1 and B7H3-C2.

[0423] Example 26 (3.1.5) Endocytosis activity test of anti-B7H3 antibody The intensity of endocytosis of anti-B7H3 antibody in A549 human non-small cell lung cancer cells was detected by FACS method. A549 cells grown to logarithmic growth phase were digested with trypsin, centrifuged to collect the cells, and washed three times with pre-cooled PBS. The cells were resuspended in PBS (containing 1% BSA), and the test anti-B7H3 antibodies were added at a concentration of 10 μg / mL, and incubated at 4°C for 1 hour. The cells were collected by centrifugation, washed three times with PBS, and resuspended in DMEM + 10% FBS. After resuspension, the cells were divided into four portions and incubated at 37°C for 0, 1, 2, and 4 hours, respectively. After incubation was completed, the cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μL of 1% BSA (in PBS), and 1 μL of fluorescent secondary antibody APC anti-human IgG (Biolegend) was added to each well, mixed evenly, and incubated at 4°C for 0.5 hours. The cells were collected by centrifugation, washed three times with pre-chilled PBS, and resuspended in 200 μL of PBS and detected by the instrument. The formula was: Endocytosis rate (%) = [1-(mean fluorescence value of test sample at the time point-mean fluorescence value of negative control sample at the time point) / (mean fluorescence value of test sample at 0 hours-mean fluorescence value of negative control sample at 0 hours)] × 100. The results of 1D1-01 and 2E3-02 are shown in Figures 6A and 6B, respectively, and the anti-B7H3 antibodies 1D1-01 and 2E3-02 showed strong endocytosis activity in A549 tumor cells, reaching about 40% at 4 hours, which was comparable to antibody B7H3-C1.

[0424] 4. Coupling of antibodies with compounds containing cellular biologically active molecules and linkers Example 27 (4.1): Preparation of B7H3-ADC-01 0.3mL of B7H3 antibody (2E3-02, 17mg / mL) was taken and diluted with 0.13mL of 20mM PB+150mM NaCl+20mM sodium edetate solution (pH7.6), then 0.23mL of 20mM PB+150mM NaCl solution (pH7.6) was added and mixed uniformly, the pH was adjusted to 7.4 with 1M K2HPO4 solution, 10mM TCEP (tris(2-carboxyethyl)phosphine, 0.017mL, 0.17μmol) solution was added and mixed uniformly, and the mixture was left at room temperature for 30 minutes. A dimethylsulfoxide (0.1mL) solution of DL-001 (1.0mg, 20 times the molar amount of the antibody substance) was added to the above solution reaction system, mixed uniformly, and left at room temperature for 2 hours, after which 6.1μl of 100mM cysteine ​​was added to stop the reaction. Finally, the buffer was replaced with 20 mM PB buffer at pH 6.4 using a G-25 gel column. The coupling product B7H3-ADC-01 (DAR8) of DL-001 and B7H3 antibody was obtained. The DAR value measured by mass spectrometry was 8.0.

[0425] [ka]

[0426] The method for measuring the DAR value by mass spectrometry is as follows. Chromatography conditions: Chromatography column: PLRP-S, 2.1 × 50 mm, 5 μm; Mobile phase A: 0.1% FA / HO; Mobile phase B: 0.1% FA / ACN Column temperature: 30°C; Sample chamber temperature: 8°C; Flow rate: 0.6 ml / min; Flow rate: 2 μl [Table 13] Sample treatment: 50 μg of ADC-01 sample was taken, 2 μl of 1M DTT was added, and 50 μl of the mixture was diluted with ultrapure water to a concentration of about 1.0 mg / ml, mixed uniformly, and reduced at room temperature for 30 minutes.

[0427] LC / MS model: Agilent 1290-6545XT Q-TOF.

[0428] Mass spectrometry conditions: gas temperature: 320°C, drying gas: nebulizer: 35 psi; sheath gas temperature: 350°C; sheath gas flow rate: 11 l / min; m / z 500-3000. [Table 14]

[0429] In the table, LC represents the light chain of the antibody, HC represents the heavy chain of the antibody, DAR1 represents a complex containing a light chain or a heavy chain coupled to one payload molecule, DAR2 represents a complex containing a light chain or a heavy chain coupled to two payload molecules, and DAR3 represents a complex containing a light chain or a heavy chain coupled to three payload molecules. Hereinafter, LC, HC, DAR1, DAR2, and DAR3 are as described above.

[0430] It was found that the light chain of the 2E3-02 antibody was coupled to one payload molecule (DAR1 ratio was 100.0%) and the heavy chain was coupled to three payload molecules (DAR3 ratio was 100%), so the antibody-drug conjugation ratio (DAR value) of B7H3-ADC-01 was calculated to be 8.0. Since the light chain was coupled to 100% of the DAR1 position of one payload molecule and the heavy chain was coupled to three payload molecules (DAR3 ratio was 100%), q was found to be 8.

[0431] Example 28 (4.2): Preparation of B7H3-ADC-02 Using a procedure similar to that for B7H3-ADC-01, DL-001 was replaced with DL-002 to obtain the coupling product of DL-002 and the B7H3 antibody 2E3-02, B7H3-ADC-02 (DAR8), with a DAR value of 8.0 as determined by mass spectrometry.

[0432] [ka]

[0433] Example 29 (4.3): Preparation of B7H3-ADC-03 Using the same procedure as for B7H3-ADC-01, the amount of DL-001 added was reduced from 20 times the molar amount of the antibody substance to 4.4 times the molar amount of the antibody substance to obtain B7H3-ADC-03 (DAR4.2), which is a coupling product of DL-001 and B7H3 antibody 2E3-02. The DAR value measured using the same method as in Example 4.1 was 4.2.

[0434] [ka]

[0435] [Table 15]

[0436] The light chain of the 2E3-02 antibody was found to be coupled with 0 to 1 payload molecules (LC and DAR1 ratios were 76.0% and 24.0%, respectively), and the heavy chain was coupled with 0 to 3 payload molecules (HC, DAR1, DAR2, DAR3 ratios were 12.0%, 21.0%, 36.0%, and 31.0%), thereby calculating the antibody-drug conjugation ratio (DAR value) of B7H3-ADC-03 to be 4.2. From the distribution of coupled payload molecules on the heavy and light chains, it was found that q could be 1, 2, 3, 4, 5, 6, 7, or 8.

[0437] Example 30 (4.4): Preparation of Her2-ADC-04 Using the same procedure as for B7H3-ADC-01, the antibody was replaced with Trastuzumab, and DL-001 was replaced with DL-002 to obtain Her2-ADC-04, which is a coupling product of DL-002 and Trastuzumab. The DAR value measured using the same method as in Example 4.1 was 7.9.

[0438] [ka] [Table 16]

[0439] It was found that the light chain of the Trastuzummab antibody was coupled with 0-1 payload molecules (LC and DAR1 ratios were 3% and 97%), and the heavy chain was coupled with 0-4 payload molecules (HC, DAR1, DAR2, DAR3, and DAR4 ratios were 0%, 0%, 6%, 90%, and 4%, respectively), thereby calculating the antibody-drug conjugation ratio (DAR value) of Her2-ADC-04 to be 7.9. From the distribution of coupled payload molecules on the light chain, it was found that q could be 2, 3, 4, 5, 6, 7, 8, or 10.

[0440] Example 31 (4.5): Preparation of Reference ADC-01 The preparation of Reference DL was performed in accordance with the literature (WO2017151979), and its structure is as shown below.

[0441] [ka]

[0442] Using the same procedure as in Example 4.4, DL-002 was replaced with reference DL to obtain reference ADC-01, as shown below: The measured DAR value was 7.4.

[0443] [ka]

[0444] 5. Detection of the inhibitory activity of ADC against in vitro cell activity Example 32 (5.1): Detection of inhibitory activity of ADC against in vitro cellular activity Using conventional methods, HT29, NCI-H358 (HT29 and NCI-H358 are B7H3 positive tumor cells), SW480, A375, A549 and Calu-3 (Calu-3 is B7H3 negative tumor cells) tumor cells were digested with trypsin, the cells in the tubes were collected, suspended in the corresponding test medium (containing 2% FBS), and added to a 96-well plate at 2000-5000 cells / well. 100uL of ADC diluted with 2% FBS medium was added to the 96-well plate, with a starting concentration of 150μg / ml, and diluted 3-fold (12 concentration gradient). After incubation at 37℃ 5%CO2 for 7 days, 20μL of CCK8 reagent was added to each well, reacted for 2-6 hours, and read by a microplate reader (detection wavelength: 450nm). The test results are shown in Table 3-1, Table 3-2 and Figure 7A-E.

[0445] The test results showed that the ADC molecules of the present disclosure have a killing effect on tumor cells. On the other hand, when the ADC molecules of the present disclosure were tested against B7H3-negative cells Calu-3, the killing effect was shown to be weak (IC 50 >10 7 ng / ml), indicating that the ADC molecules of the present disclosure have excellent selective targeted killing effects against B7H3 positive and negative tumor cells in vitro.

[0446] Table 3-1. Cell inhibitory activity test of B7H3-ADC-01 [Table 17] Table 3-2. Inhibitory activity test of Her2-ADC-04 against cells [Table 18]

[0447] Example 33 (5.2): Efficacy test of ADC against NCI-HT29 transplanted tumors 1. Experimental Materials Test compound: B7H3-ADC-01 Control compound: B7H3 monoclonal antibody (2E3-02) Experimental cells: NCI-HT29 cells, purchased from ATCC; Experimental animals: Balb / c nu nude mice, female, 5-6 weeks old, purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0448] 2. Experimental Plan 2.1. Cell treatment NCI-HT29 cells were cultured in 15 cm diameter culture dishes in 1640 medium containing 10% FBS. When the cell density reached approximately 80–90%, they were digested with trypsin-EDTA, washed twice with PBS, centrifuged, resuspended in pre-chilled PBS, counted in a cell counter, and diluted with PBS to a cell concentration of 5 × 10 7 / mL.

[0449] Tumor cell transplantation Balb / c nu mice were acclimated to the laboratory environment for 2–5 days, and then subcutaneously inoculated with NCI-HT29 cells into the right rib cage. The cell volume was 5 × 10 6 The inoculation volume was 0.2 mL (containing 50% Matrigel) per mouse. 3 The experiment was carried out when the plants had grown to .

[0450] 2.3. Animal treatment and testing Groups of tumor-bearing nude mice were administered the following regimen: [Table 19] After the end of the administration cycle, the mice were continuously observed for 1 to 2 weeks. After the final measurement of the tumor volume, the tumors were isolated, accurately weighed, and photographed for record keeping.

[0451] 2.4. Measurement of tumor volume and body weight: Tumor volumes and body weights were measured on days 1, 4, 8, 11, 15, and 18 of the experiment, and TGI% was calculated.

[0452] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 長さ ×L 短さ 2 .

[0453] The formula for calculating TGI was TGI(%)=(1-T / C)×100%, where T and C are the relative tumor volumes at a particular time point in the treatment and control groups, respectively.

[0454] 3. Experimental Results Table 3-3. NCI-HT29 transplanted tumor model data (tumor volume mm 3 ) [Table 20] The test results show that the ADC of the present application has a very strong tumor suppression effect. During the administration period, no significant weight loss or significant drug toxicity was observed in any animal group. The detection results are shown in Table 3-3 and Figure 8A-B.

[0455] Example 34 (5.3), ADC Plasma Stability Study Solution preparation: Test ADC stock solutions were diluted in PBS to a working solution concentration of 2 mg / mL.

[0456] Cultivation of samples: B7H3-ADC-01, B7H3-ADC-03, Her2-ADC-04, reference ADC-01, and human plasma were all added with the bactericide ProClin to a final concentration of 0.1%, and were operated in a sterile environment. B7H3-ADC-01, B7H3-ADC-03, Her2-ADC-04, and reference ADC-01 were each added to the above sterile plasma at a final concentration of 200ug / ml, and cultured in a cell incubator at 37°C with a shaking speed of 80rpm. After culturing for 0 days and 14 days, the ADC samples were taken out, 100ul of protein A was added to each tube, adsorbed for 2 hours, and eluted to obtain the cultured ADC. The DAR value of the cultured ADC (detection and calculation methods are the same as in Example 4.1) was detected, and the plasma stability of the sample was measured. The detection results are shown in Table 3-4.

[0457] Table 3-4 Plasma stability studies of ADCs [Table 21]

[0458] The test results showed that the DAR values ​​of B7H3-ADC-01, B7H3-ADC-03 and Her2-ADC-04 were substantially maintained during plasma culture (i.e., the payload linker was hardly separated from the antibody), while the decrease in DAR of the reference ADC-01 with a maleimide linker was very significant after 14 days of culture. The experimental results demonstrated that all the ADCs of the present invention have better plasma stability compared to the reference ADC-01.

[0459] Although the specific embodiments of the present invention have been described above, those skilled in the art will recognize that these are merely illustrative and may make various changes and modifications to these embodiments without violating the principles and substance of the present invention. Therefore, the scope of protection of the present invention is limited by the appended claims.

Claims

1. A conjugate of antibody-eribulin or a derivative thereof represented by formula I, or a stereoisomer of said conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof. 【Chemistry 1】 (however, Tb is an antibody or antigen-binding fragment thereof bound to a target, or a targeting moiety; q is the drug-Tb coupling ratio; R １ , R ２ are each independently selected from hydrogen, deuterium, and C1-6 alkyl; or R １ and R ２ form a 5- to 6-membered heterocycle together with the nitrogen atom and oxygen atom to which they are linked, said 5- to 6-membered heterocycle being optionally substituted by one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, and C3-6 heterocyclyl; L １ teeth, 【Chemistry 2】 wherein position 1 is linked to Tb via an S atom and position 2 is selected from L ２ is connected to R ｃ is selected from hydrogen, C1-6 alkyl; L ２ teeth, 【Chemistry 3】 and position 1 is selected from L １ and position 2 is L ３ is connected to p is selected from any integer between 0 and 10; Y is -CH ２ --, --OCH ２ CH ２ - selected from Z is -CR ３ R ４ --, --NR ５ - selected from R ３ , R ４ are each independently selected from hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C3-6 heterocyclyl, or R ３ and R ４ form a 3- to 6-membered carbocyclic or 3- to 6-membered heterocyclic ring together with the carbon atom to which they are attached, R ５ is selected from hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C3-6 heterocyclyl; L ３ is a chemical bond, 【Chemistry 4】 , amino acid residues or short peptides consisting of 2 to 10 amino acid residues, said amino acid residues being selected from natural amino acid residues, non-natural amino acid residues or AA １ or a stereoisomer thereof, A.A. １ The structure of the amino acid residue represented by is as shown below. 【Chemistry 5】 however, R ａ , R ｂ are each independently hydrogen and 【Chemistry 6】 and R ａ , R ｂ is not hydrogen at the same time, Or, R ａ and R ｂ together with the carbon atom to which they are attached form a 4- to 10-membered heterocyclic ring, said 4- to 10-membered heterocyclic ring optionally containing one or more R ０ is replaced by r,r １ are each independently selected from any integer between 0 and 20; R ｍ１ , R ｎ１ are each independently selected from hydrogen, C1-6 alkyl, and C3-6 cycloalkyl; Or, R ｍ１ and R ｎ１ together with the nitrogen atom to which they are attached form a 4- to 10-membered heterocycle, said 4- to 10-membered heterocycle optionally containing one or more R ０’ is replaced by R ０ , R ０’ are each independently C1-6 alkyl, C3-6 cycloalkyl, -NR ｍ２ R ｎ２ and 4-10 membered heterocyclyl optionally substituted with C1-6 alkyl; R ｍ２ , R ｎ２ are each independently selected from hydrogen and C1-6 alkyl; L ４ is absent or present, L ４ If there is L ４ teeth, 【Chemistry 7】 and position 1 is selected from L ３ and position 2 is linked to a nitrogen atom of eribulin; Also, L １ but 【Chemistry 8】 where position 1 is linked to Tb via an S atom and position 2 is L ２ When linked to L ３ is not a chemical bond, Val-Cit, Val-Lys, Gly-Gly-Phe-Gly or Ala-Ala-Asn, or L ４ Although there exists 【Chemistry 9】 isn't it.)

2. (1) Tb is an antibody or an antigen-binding fragment thereof; (i) the antibody or antigen-binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity determining region fragment, a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a probody, a monoclonal antibody, a bispecific antibody, or a multispecific antibody; (ii) Tb is a monoclonal antibody or an antigen-binding fragment thereof; (iii) Tb is an antibody or antigen-binding fragment thereof having endocytosis activity; (iv) Tb is an antibody or an antigen-binding fragment thereof having an activity of binding to a free antigen in tumor tissue and / or a tumor cell surface antigen; (v) Tb, growth factor, integrin α5β6, integrin α4β7, 0772P, 5T4, ACTA2, ADGRE1, AGS-16, AIF1, AKR1C1, AKR1C2, ANGPTL4, ApoE, ASLG659, BAFF-R, BMPR1B, BNIP3, Brevican, C1QA, C1QB, CA6, pCAD, P-cadherin, CADM1, CCL5, CCR5, CDllb, CD1 lc, CD19, CD20, CD21, CD22, CD30, CD33, CD37, CD40, CD45 (PTPRC), CD49D (ITGA4), CD56, CD66e, CD70, CD7 2. CD74, CD79a, CD80, CD138, CD2 23. CDCP1, CDH11, Claudin18.2, COL6A3, COL7A1, CRIPTO, CSF1R, C TGF、CTSD、CTSS、CXCL10、CXCL11 , CXCR5, DDIT4, DLL3, DLL4, DR5, E16、EFNA4、B7H3、EGFR、EGFRvII I, EGLN, EGLN3, EMR2, Endothelin receoptor, ENPP3, EpCAM, EphB2R, ETBR , FGF2, FGFR2, FcRH1, FcRH2, GEDA, GPNM B、GZMB、Her2、Her3、HLA-DOB、HMOX1、IF NG, IFI6, IGF-1R, IGFBP3, IL10RA1, IL20 Rα, IL-6, IRTA2, KISS1R, KRT33A, LOX, L RRC15, LUM, LY6E, LY64, LY86, LYPD3, MCP T8, MDP, Mesothelin, c-Met, MFI2, MMP1 0. MMP14, MMP16, MPF, MSG783, MS4A7, MUC 1. MUC16, NaPi2b, NaPi3b, NCA, Nectin 4. NOG, P2X5, PD-L1, PDK1, PDGFRA, PFKFB3, P GF、PGK1、PIK3AP1、PIK3CD、PLOD2、PSCA、PSCA hlg,PSMA,RNF43,ROR1,Sema5b,SERPINE1,SLC39A6,SLITRK6,SLTRK6,STAT1,STC2,STEAP1,STEAP2,TCF4,TENB2,TGF,TGFBR1,TGFB2,Tissuefactor), TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, and WNT5A; (vi) Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-Trop-2 antibody or antigen-binding fragment thereof, an anti-Her2 antibody or antigen-binding fragment thereof, an anti-Her3 antibody or antigen-binding fragment thereof, or an anti-EGFR antibody or antigen-binding fragment thereof; (2) the condition that q is selected from any number between 0.1 and 16.0, preferably, q is selected from any number between 1 and 10, more preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, and 8; (3) The above R １ , R ２ is hydrogen, or R １ and R ２ together with the nitrogen and oxygen atoms connected to them. 【Chemistry 10】 and position 1 is L ４ Or L ３ Conditions linked to (4) Said L １ teeth, 【Chemistry 11】 wherein position 1 is linked to Tb via an S atom and position 2 is selected from L ２ is connected to Preferably, L １ teeth, 【Chemistry 12】 wherein position 1 is linked to Tb via an S atom and position 2 is selected from L ２ More preferably, L １ teeth, 【Chemistry 13】 where position 1 is linked to Tb via an S atom and position 2 is L ２ Conditions linked to (5) The above R ｃ is hydrogen, (6) The condition that p is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; (7) The Z is -CH ２ -, -C(CH ３ ) ２ - or -NH-, (8) The R ３ , R ４ are each independently selected from hydrogen and methyl; (9) The R ５ is H or methyl; (10) Said L ３ are the amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, and AA. １ or 2 to 10 of Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AA １ a short peptide consisting of amino acid residues selected from Preferably、L ３ ー １ 、&--Cit、Cit-&l、Cit-Ala、&l-Ala、Ly--&、&--Lys(Ac) １ 、Ala-AA １ ,Gly-AA １ ,AA １ - １ -Gly、Ala-AA １ -Gly、Gly-AA １ -Gly、Lys-(Ala) ２ -Asn、Lys-(Ala) ２ -Asp、Gly-Phe-Gly、(Gly) ２ -Phe-Gly、D-Vl-Leu-Lys、Gly-Gly-Arg、Al-Alm-Ae、G-------- ２ 、Val-Lys、Lys-Ala-Asn、(Gly) ２ -Phe-AA １ from selected More preferably, L ３ is Val-AA １ -Gly, Val-AA １ , Val-Cit, (Gly) ２ -Phe-AA １ Particularly preferably, L ３ is Val-AA １ , Val-Cit; (11) The R ａ , R ｂ Among these, any one is hydrogen and the other is 【Chemistry 14】 and Or, R ａ and R ｂ R together with the carbon atom connected thereto ０ and preferably R ａ and R ｂ together with the carbon atoms connected to them 【Chemistry 15】 The first carbon atom is R ａ and R ｂ the condition that the carbon atom is connected with (12) The r and r １ are each independently selected from 0, 1, 2, 3, 4 and 5, preferably r, r １ are each independently selected from 0 and 4, more preferably, r is 0 and r １ is 4, (13) The R ｍ１ , R ｎ１ are each independently selected from C1-6 alkyl; or R ｍ１ and R ｎ１ may optionally be R ０’ forming a 5- to 6-membered heterocyclic ring substituted by Preferably, R ｍ１ , R ｎ１ are each independently selected from methyl, ethyl, n-propyl, and n-butyl; or R ｍ１ and R ｎ１ may optionally be R ０’ conditions for forming a piperidine or piperazine ring substituted with (14) The R ０ , R ０’ are each independently C1-6 alkyl, -NR ｍ２ R ｎ２ and 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl, (15) The R ｍ２ , R ｎ２ is methyl, (16) Said L ２ teeth, 【Chemistry 16】 and position 1 is selected from L １ and position 2 is L ３ Conditions linked to The complex of claim 1, or a stereoisomer of said complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, characterized in that it satisfies one or more of the following:

3. (1) L ２ teeth, 【Chemistry 17】 and position 1 is selected from L １ and position 2 is L ３ is connected to Preferably, L ２ teeth, 【Chemistry 18】 and position 1 is L １ and position 2 is L ３ Conditions linked to (2) L ３ is a chemical bond, 【Chemistry 19】 and position 1 is selected from L ２ and position 2 is L ４ or linked to a nitrogen atom of eribulin, Preferably, L ３ teeth, 【Chemistry 20】 and position 1 is selected from L ２ and position 2 is L ４ or linked to a nitrogen atom of eribulin, More preferably, L ３ teeth, 【Chemistry 21】 and position 1 is selected from L ２ and position 2 is L ４ or linked to a nitrogen atom of eribulin, Particularly preferably, L ３ teeth, 【Chemical 22】 and position 1 is selected from L ２ and position 2 is L ４ or linked to the nitrogen atom of eribulin, (3) AA １ The amino acid residue represented by 【Chemistry 23】 is selected from Preferably, AA １ The amino acid residue represented by 【Chemistry 24】 is selected from More preferably, AA １ The amino acid residue represented by 【Chemistry 25】 The condition that (4) L ４ is absent or present, L ４ If there is L ４ teeth, 【Chemistry 26】 and position 1 is selected from L ３ and position 2 is linked to a nitrogen atom of eribulin; Preferably, L ４ teeth, 【Chemical 27】 and position 1 is L ３ and position 2 is linked to the nitrogen atom of eribulin; 3. The complex according to claim 1 or 2, or a stereoisomer of said complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable solvate thereof, characterized in that it satisfies one or more of the following:

4. Linker, 【Chemistry 28】 is selected from the formula: 【Table 1】 Where Y, Z, p, and AA １ is as defined in any one of claims 1 to 3, wherein position 1 is linked to Tb via an S atom and position 2 is linked to the nitrogen atom of eribulin; Preferably, the linker 【Chemical 29】 is selected from the formula: 【Table 2】 Where Y, Z, p, and AA １ is as defined in any one of claims 1 to 3, wherein position 1 is linked to Tb via an S atom and position 2 is linked to the nitrogen atom of eribulin; More preferably, the linker 【Chemistry 30】 is selected from the formula: 【Table 3】 Where Y, Z, p, and AA １ is as defined in any one of claims 1 to 3, wherein position 1 is linked to Tb via an S atom and position 2 is linked to a nitrogen atom of eribulin; The complex according to any one of claims 1 to 3, or a stereoisomer of the complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

5. Linker 【Chemistry 31】 is selected from the formula: 【Table 4-1】 【Table 4-2】 【Table 4-3】 Position 1 is linked to Tb via a S atom, and position 2 is linked to the nitrogen atom of eribulin; Preferably, the linker 【Chemistry 32】 is selected from the formula: 【Table 5-1】 【Table 5-2】 Position 1 is linked to Tb via a S atom, and position 2 is linked to the nitrogen atom of eribulin; More preferably, the linker 【Chemical 33】 is selected from the formula: 【Table 6】 Position 1 is linked to Tb via an S atom, and position 2 is linked to the nitrogen atom of eribulin; The complex according to any one of claims 1 to 4, or a stereoisomer of the complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

6. The antibody-eribulin or derivative thereof conjugate according to any one of claims 1 to 5, characterized in that the conjugate is selected from the following: a stereoisomer of the conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof. 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical 38】 【Chemical 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】

7. Tb is (i) Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, and the anti-Her2 antibody is ambenitamab, coprelotamab, disitamab, gancotamab, mergetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab, or an antigen-binding fragment thereof; (ii) the scheme in which Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof, and the anti-Trop-2 antibody is datopotamab, sacituzumab, or an antigen-binding fragment thereof; (iii) Tb is an anti-EGFR antibody or an antigen-binding fragment thereof, and the anti-EGFR antibody is demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serculutamab, tomuzotuximab, zalutumumab, cetuximab, or an antigen-binding fragment thereof; (iv) the scheme in which Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody is enoblitzumab, mirzotamab, omburtamab, 1D1-01, 2E3-02 antibody or an antigen-binding fragment thereof; (v) Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, wherein said anti-B7H3 antibody or antigen-binding fragment thereof comprises the complementarity determining regions (CDRs) of HCDR1 having the sequence SEQ ID NO:9, HCDR2 having the sequence SEQ ID NO:10, HCDR3 having the sequence SEQ ID NO:11; and / or LCDR1 having the sequence SEQ ID NO:12, LCDR2 having the sequence SEQ ID NO:13, LCDR3 having the sequence SEQ ID NO:14, wherein the CDRs are numbered according to the scheme defined by the Kabat numbering system; (vi) Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, wherein said anti-B7H3 antibody or antigen-binding fragment thereof comprises the complementarity determining regions (CDRs) of HCDR1 having the sequence SEQ ID NO:15, HCDR2 having the sequence SEQ ID NO:16, HCDR3 having the sequence SEQ ID NO:17; and / or LCDR1 having the sequence SEQ ID NO:18, LCDR2 having the sequence GAS, LCDR3 having the sequence SEQ ID NO:19, wherein the CDRs are numbered according to the scheme defined by the IMGT numbering system; (vii) Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO: 1 or 5, and / or a VL set forth in SEQ ID NO: 2 or 6; Furthermore, (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof; and / or (b) a scheme comprising a human immunoglobulin light chain constant region (CL) or a variant thereof; (viii) Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:5, a CH set forth in SEQ ID NO:20 or a variant thereof, and / or a VL set forth in SEQ ID NO:6, a CL set forth in SEQ ID NO:21 or a variant thereof; Preferably, the B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:3 and / or a light chain as set forth in SEQ ID NO:4; or a heavy chain as set forth in SEQ ID NO:7 and / or a light chain as set forth in SEQ ID NO:

8. The complex according to any one of claims 1 to 6, or a stereoisomer of the complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, characterized in that the complex is any one of:

8. The complex is selected from any one of the following formulas: 【Chemistry 46】 with the proviso that B7H3-mAb is the 2E3-02 antibody, and q is selected from any integer between 0.1 and 10.0, preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8; 【Chemistry 47】 wherein q is selected from any integer between 0.1 and 10.0, and preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8; 【Chemistry 48】 with the proviso that the B7H3-mAb is the 2E3-02 antibody and q is selected from any integer between 0.1 and 10.0, preferably q is selected from 1, 2, 3, 4, 5, 6, 7 and 8; or 【Chemistry 49】 wherein q is selected from any integer between 0.1 and 10.0, and preferably q is selected from 1, 2, 3, 4, 5, 6, 7, and 8; The complex according to any one of claims 1 to 7, or a stereoisomer of the complex, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

9. A drug linker conjugate represented by formula II, or a stereoisomer of said drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof. 【Chemistry 50】 (however, L １ but 【Chemistry 51】 If, position 1 is linked to Lg, position 2 is linked to L ２ is connected to Lg is a leaving group when reacting with Tb, L ２ , L ３ , L ４ , R ｃ , R １ and R ２ is as defined in any one of claims 1 to 6, L １ but 【Chemistry 52】 If Lg-L １ teeth, 【Chemistry 53】 And L ２ , L ３ , L ４ , R ｃ , R １ and R ２ is as defined in any one of claims 1 to 6.

10. The Lg is a halogen, a sulfonyl, a tertiary amine (Me ３ N ＋ , Et ３ N ＋ ), diazonium, -OMs, MeSO ２ -, CF ３ SO ３ -, preferably Lg is selected from F, Cl, MeSO ２ -, more preferably Lg is selected from F, MeSO ２ The drug linker conjugate of claim 9, or a stereoisomer of said conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable solvate thereof, characterized in that said stereoisomer is selected from the following:

11. The drug linker conjugate described in claim 9 or 10, characterized in that the drug linker conjugate is selected from the following formulas, or a stereoisomer of the drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof. 【Chemical 54】 【Chemistry 55】 【Chemistry 56】 【Chemistry 57】 【Chemistry 58】 【Chemistry 59】

12. A linker of formula III: 【Chemistry 60】 (Lg, L １ , L ２ , L ３ and L ４ is as defined in any one of claims 1 to 11, and Lg1 is a leaving group when reacting with eribulin or a derivative thereof, preferably Lg1 is -OH, 【Chemistry 61】 or halogen.)

13. A method for preparing a conjugate represented by formula I, comprising coupling a sulfhydryl on Tb with a drug-linker conjugate represented by general formula II. 【Chemistry 62】 (where Tb, q, Lg, L １ , L ２ , L ３ and L ４ , R １ and R ２ is as defined in any one of claims 1 to 11.

14. 14. The method of claim 13, comprising the step of coupling a sulfhydryl on Tb with the drug-linker conjugate represented by general formula II under suitable solvent and conditions to form a C-S bond. 【Chemistry 63】 (where Tb, q, Lg, L １ , L ２ , L ３ and L ４ , R １ and R ２ is as defined in any one of claims 1 to 11, The molar ratio of sulfhydryls on the Tb to the drug-linker conjugate can be 1:(1-20), e.g., 1:(2-20), 1:(4-20), 1:(6-20), 1:(8-20), 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20), or 1:(18-20); The coupling reaction is preferably carried out in water and / or an organic solvent, the organic solvent being preferably one or more of N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, nitrile-based solvents (e.g., acetonitrile) and alcohol-based solvents (e.g., methanol, ethanol); The work-up operation of the method can be a conventional work-up method of coupling reaction in the art, preferably purifying the coupling product by chromatography, the chromatographic method is usually one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography.

15. A group of conjugates of antibody-eribulin or a derivative thereof, comprising the conjugate of antibody-eribulin or a derivative thereof according to any one of claims 1 to 8, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, The antibody-eribulin or derivative thereof conjugate in the group of antibody-eribulin or derivative thereof conjugates has two or more q values, Optionally, a drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from an integer or decimal number between 1 and 10; Preferably, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from integers or decimals from 1 to 8; Preferably, the drug-antibody ratio (DAR) in the antibody-eribulin or derivative thereof conjugates is selected from integers or decimals from 2 to 8; Preferably, the drug-antibody ratio (DAR) in the group of antibody-eribulin or derivative thereof conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.2, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.

16. A pharmaceutical composition comprising a conjugate of an antibody-eribulin or a derivative thereof described in any one of claims 1 to 8, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or a drug linker conjugate represented by formula II described in any one of claims 9 to 11, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or a group of conjugates of an antibody-eribulin or a derivative thereof described in claim 15, and optionally one or more pharmaceutical auxiliary agents.

17. Use of a conjugate of an antibody-eribulin or a derivative thereof according to any one of claims 1 to 8, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or a drug linker conjugate represented by formula II according to any one of claims 9 to 11, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or a mixture according to claim 15, or a pharmaceutical composition according to claim 16, in the manufacture of a medicament for treating and / or preventing a disease associated with abnormal cellular activity, Optionally, the disease associated with abnormal cell activity is a hematological and / or solid tumor, e.g., selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, liver cancer, kidney cancer, urothelial cancer, solid tumor, non-Hodgkin's lymphoma, central nervous system tumor (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, or thyroid cancer.

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