A preparation method and use of a dual drug-linker for ADC

Abstract

The present invention discloses a method for preparing a dual-drug linker for ADC and its use. Specifically provided is a dual-drug linker assembly unit shown in formula III, or a stereoisomer thereof, or an optical isomer thereof. The dual-drug linker assembly unit can be linked with a targeting linker to obtain a dual-drug targeting linker-drug conjugate shown in formula I. The specific structure of the present invention can effectively reduce the aggregation of the dual-drug targeting linker-drug conjugate, which is beneficial to process amplification, thereby improving the efficiency of targeting on tumor cells, reducing toxic side effects on normal cells, and effectively overcoming drug resistance, thereby achieving a synergistic anti-tumor effect. The dual-drug linker assembly unit and the dual-drug targeting linker-drug conjugate of the present invention have broad application prospects in the preparation of anti-tumor drugs for prevention and / or treatment. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a preparation method of a dual-drug-linker for ADC and a use thereof. Background Art

[0002] Antibody-Drug Conjugate (ADC) can selectively deliver drugs to cancer cells and kill them, but has little effect on normal cells, creating a new era of tumor treatment. As ADC, several drugs have been approved by the FDA for marketing, such as Mylotarg, which is a CD33 antibody linked to calicheamicin, Adcetris, which is a CD30 antibody linked to auristatin E, for the treatment of patients with Hodgkin's lymphoma and undifferentiated large cell lymphoma, DS-8201, which is a Her2 antibody linked to a camptothecin derivative Dxd, for the treatment of patients with HER2-positive breast cancer, and Sacituzumabgovitecan, which targets the TROP-2 antigen (also known as epithelial glycoprotein 1, EGP-1).

[0003] So far, the drugs contained in ADCs approved by the FDA mainly target DNA or tubulin. The ADCs that have been marketed are connected by antibodies and drugs targeting DNA or tubulin, respectively. Currently, there are no ADCs that simultaneously connect antibodies and drugs targeting DNA and tubulin on the market. It is not clear whether drugs targeting DNA and tubulin can kill tumors through two different anti-tumor mechanisms and whether they can work effectively.

[0004] As small molecule anti-tumor compounds, camptothecin derivatives, such as SN-38, Dxd, and Dx-8951, which are known to inhibit DNA topoisomerase I and achieve anti-tumor effects, have been confirmed to have killing effects on various cancer cells both in vivo and in vitro, showing strong anti-tumor effects. Compounds that inhibit microtubules to achieve anti-tumor effects, such as Eribulin, MMAE, MMAF, and Maytansine, have been confirmed to have killing effects on various cancer cells both in vivo and in vitro, showing strong anti-tumor effects. Connecting two anti-tumor drugs with different mechanisms to the same antibody may have a synergistic effect on anti-tumor activity, or it may produce an antagonistic effect on anti-tumor activity, and the actual effect cannot be predicted.

[0005] Therefore, it is urgent to develop an efficient and safe ADC with multiple drug and multi-target action mechanisms, which is of great significance for the development of anti-tumor drugs with excellent anti-tumor effects and safety. Summary of the invention

[0006] The object of the present invention is to provide a dual-drug linker assembly unit and its corresponding dual-drug targeting linker-drug conjugate.

[0007] The present invention provides a dual-drug link assembly unit shown in Formula III, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof:

[0008]

[0009] Wherein, T is a tethering group, which can be connected to a targeting linker; the targeting linker is a substance that can target and bind to the lesion site;

[0010] U is a three-pronged linker part, and the structure of U is Wherein, Y1, Y2, and Y3 are each independently selected from CONH, NHCO, CO, NH, COO, OCO, or none; L a , L b , L c , L d , L e , L f , L g , L h Each independently selected from 0 to 8 methylene groups; A is selected from N, substituted or unsubstituted groups: aryl, heteroaryl, chain alkyl, condensed cycloalkyl, heterocondensed cycloalkyl, saturated cycloalkyl or saturated heterocycloalkyl, and the substituents are each independently selected from halogen, cyano, hydroxyl, C 1～6 Alkyl or C 1～6 Alkoxy;

[0011] m, n, and p are each independently selected from integers of 0 to 30, and n and p are not 0 at the same time;

[0012] W1, W2, and W3 are each independently selected from methylene, Alkenylene, alkynylene, 3- to 8-membered aryl, 3- to 8-membered heteroaryl; Wa is selected from an integer of 2 to 8;

[0013] L1 and L2 are cleavable or non-cleavable linking groups;

[0014] D1 and D2 are the first drug structural unit and the second drug structural unit, respectively. The structures of D1 and D2 are the same or different.

[0015] Furthermore, the T can react and connect with the sulfhydryl group or the amino group on the targeting linker.

[0016] Furthermore, the The structure is selected from:

[0017]

[0018]

[0019]

[0020] Furthermore, the structure of the dual-drug linkage assembly unit is shown in Formula IV:

[0021]

[0022] Among them, the structure of U is Wherein, Y1, Y2, and Y3 are each independently selected from CONH, CO, NH, O, or none; L a , L b , L c , L d , L e , L f , L g , L w , L v Each independently selected from 0 to 4 methylene groups; A is selected from N, substituted or unsubstituted groups: aryl, heteroaryl, chain alkyl, saturated cycloalkyl or saturated heterocycloalkyl, and the substituents are each independently selected from halogen, cyano, hydroxyl, C 1～6 Alkyl or C 1～6 Alkoxy;

[0023] m, n, and p are each independently selected from integers of 0 to 30, and n and p are not 0 at the same time;

[0024] W1, W2, W3 are each independently selected from methylene, Alkenylene, alkynylene, 3- to 8-membered aryl, 3- to 8-membered heteroaryl; Wa is selected from 2 to 4 integers;

[0025] X 1 , X 2 Each independently selected wherein a, b, c, d are each independently selected from 0 or 1, R1, R2, R3, R4 are each independently selected from H, C 1～5 Alkyl, substituted or unsubstituted benzyl, -L7NHCONH2, L7 is 0 to 3 methylene groups;

[0026] B 1 , B 2 , C 1 , C 2 , E 1 , E 2 Each is independently selected from the following substituted or unsubstituted groups: L8NHL3, L4OL5 or none; the substituents are each independently selected from C 1～5 Alkyl; wherein L8, L3, L4, L5, L6 are each independently selected from 0 to 2 methylene groups;

[0027] D1 and D2 are each independently selected from cytotoxic drugs, drugs for treating autoimmune diseases or anti-inflammatory drugs;

[0028] T as described above.

[0029] Furthermore, the structure of the dual-drug linkage assembly unit is shown in Formula V:

[0030]

[0031] Wherein, Y1, Y2, and Y3 are each independently selected from CONH, CO, NH, O, or none; L a , L b , L c , L d , L e , L f , L g , L w , L v Each independently selected from 0 to 4 methylene groups; A selected from N, substituted or unsubstituted phenyl, substituted or unsubstituted The substituents are independently selected from halogen, cyano, hydroxyl, C 1～6 Alkyl or C 1～6 Alkoxy;

[0032] m, n, and p are each independently selected from integers of 0 to 30, and n and p are not 0 at the same time;

[0033] W1, W2, and W3 are each independently selected from methylene, Wa is selected from an integer of 2 to 3; and at least one of W2 and W3 is

[0034] X 1 , X 2 , B 1 , B 2 , C 1 , C 2 , E 1 , E 2 , D1 and D2 are as described above.

[0035] Furthermore, the structure of the dual-drug linkage assembly unit is shown in Formula VI-1, VI-2, VI-3 or VI-4:

[0036]

[0037]

[0038] Wherein, m, n, and p are each independently selected from integers of 0 to 30;

[0039] W1, W2, W3 are each independently selected from methylene, Wa is selected from an integer of 2 to 3; and at least one of W2 and W3 is

[0040] M is selected from halogen, cyano, hydroxyl, C 1～6 Alkyl or C 1～6 Alkoxy;

[0041] L a , L b , L c , L d , L g Each independently selected from 0 to 2 methylene groups;

[0042] X 1 , X 2 , B 1 , B 2 , C 1 , C 2 、E 1 、E 2 , D1, D2 are as described above.

[0043] Furthermore, the structure of the dual-drug linkage assembly unit is selected from one of the following structures:

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] Wherein, m is selected from an integer of 0 to 8;

[0050] n and p are each independently selected from integers of 0 to 20, and n and p are not 0 at the same time;

[0051] W1, W2, W3 are each independently selected from methylene, Wa is selected from an integer of 2 to 3; and at least one of W2 and W3 is

[0052] L a , L b , L c , L d , L g Each independently selected from none, methylene or ethylene;

[0053] D1 and D2 are as described above.

[0054] Furthermore, the structure of the dual-drug linkage assembly unit is one of the following structures:

[0055]

[0056]

[0057]

[0058] wherein n and p are independently selected from integers of 0 to 20, and n and p are not 0 at the same time; W2 and W3 are independently selected from methylene, And at least one of W2 and W3 is

[0059] L b , L d Each independently selected from none or ethylene;

[0060] D1 and D2 are as described above.

[0061] Furthermore, D1 and D2 are each independently selected from a drug unit targeting DNA or a drug unit targeting microtubules; the drug unit targeting DNA is preferably SN-38, Dxd, Dx-8951 or a derivative thereof, and / or, the drug unit targeting microtubules is preferably Eribulin, MMAE, MMAF, maytansine or a derivative thereof.

[0062] Furthermore, the dual-drug linkage assembly unit is one of the following structures:

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] The present invention also provides a dual-drug targeting linker-drug conjugate molecule, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, wherein the dual-drug targeting linker-drug conjugate molecule is obtained by connecting a targeting linker and q of the above-mentioned dual-drug linking assembly units; the targeting linker is a substance that can target and bind to a lesion site; the structure of the dual-drug targeting linker-drug conjugate molecule is shown in Formula I:

[0072]

[0073] Wherein, Ab is a targeting linker, 1≤q≤8, and T, W1, W2, W3, m, n, p, U, L1, L2, D1, and D2 are as described above.

[0074] Furthermore, the targeting linker is an antibody, an antibody fragment, a protein, a polypeptide or a nucleic acid aptamer, and the antibody is preferably an antibody against a cell surface receptor and a tumor-associated antigen.

[0075] Furthermore, the structure of the dual-drug targeting linker-drug conjugate molecule is selected from one of the following structures:

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] The present invention also provides a dual-drug targeting linker-drug conjugate, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: the dual-drug targeting linker-drug conjugate is obtained by connecting a targeting linker and the above-mentioned dual-drug linking assembly unit; the targeting linker is a substance that can target and bind to the lesion site, preferably an antibody, an antibody fragment, a protein or a nucleic acid aptamer; the antibody is preferably an antibody against cell surface receptors and tumor-associated antigens.

[0085] Further, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIa, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5±1.0, preferably 3.5;

[0086] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIb, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8±1.0, preferably 3.8;

[0087] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIc, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9±1.0, preferably 3.9;

[0088] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XId, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1±1.0, preferably 4.1;

[0089] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by Formula XIe, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.6±1.0, preferably 4.6;

[0090] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIf, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7±1.0, preferably 3.7;

[0091] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIg, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2±1.0, preferably 4.2;

[0092] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIh, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1±1.0, preferably 4.1;

[0093] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIi, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.7±1.0, preferably 4.7;

[0094] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIj, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.8±1.0, preferably 2.8;

[0095] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIk, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.9±1.0, preferably 2.9;

[0096] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIl, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5±1.0, preferably 4.5;

[0097] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIm, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.4±1.0, preferably 4.4;

[0098] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula XIn, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7±1.0, preferably 3.7;

[0099] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIo, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9±1.0, preferably 3.9;

[0100] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIp, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5±1.0, preferably 3.5;

[0101] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIq, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.8±1.0, preferably 4.8;

[0102] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula XIr, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0, preferably 4.9;

[0103] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIs, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2±1.0, preferably 4.2;

[0104] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula Xit, and the DAR value of the dual-drug targeting linker-drug conjugate is 5.3±1.0, preferably 5.3;

[0105] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula XIu, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5±1.0, preferably 4.5;

[0106] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIv, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0, preferably 4.9;

[0107] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula XIw, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8±1.0, preferably 3.8;

[0108] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by Formula XIx, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0, preferably 4.9;

[0109] Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula XIy, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.8±1.0, preferably 4.8.

[0110] The "DAR value" represents the average number of dual-drug linker assembly units coupled to a targeting linker in a dual-drug targeting linker-drug conjugate, which is equivalent to the average value of the q value. The DAR value may not be an integer.

[0111] The present invention also provides the above-mentioned dual-drug targeting linker-drug conjugate molecule, or a method for preparing the above-mentioned dual-drug targeting linker-drug conjugate, characterized in that: the method comprises the following steps: coupling the targeting linker with the dual-drug linking assembly unit to obtain.

[0112] The present invention also provides a drug for preventing and / or treating tumors, which is a preparation made of the above-mentioned dual-drug targeting linker-drug conjugate molecule or the above-mentioned dual-drug targeting linker-drug conjugate as an active ingredient and pharmaceutically acceptable excipients.

[0113] The present invention also provides the use of the above-mentioned dual-drug targeting linker-drug conjugate molecule or the above-mentioned dual-drug targeting linker-drug conjugate in preparing drugs for preventing and / or treating tumors.

[0114] Furthermore, the tumor is a HER2-positive tumor.

[0115] Furthermore, the tumor is selected from lung cancer, urethral cancer, colorectal cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumors, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma or sarcoma.

[0116] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0117] The tethering group refers to a group that can be connected to the targeting linker.

[0118] The term "substituted" herein refers to the replacement of one, two or more hydrogen atoms in a molecule by other different atoms or molecules, including one, two or more substitutions on the isotopic or ectopic atoms in the molecule.

[0119] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated π electron system, such as phenyl and naphthyl. The aryl ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent must be on a carbon atom on the ring with a conjugated π electron system. Aryl groups may be substituted or unsubstituted.

[0120] "Heteroaryl" refers to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to here include oxygen, sulfur and nitrogen. For example, furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted.

[0121] "Fused cycloalkyl" refers to a polycyclic cycloalkyl group in which two rings share two adjacent carbon atoms.

[0122] "Heterofused cyclyl" refers to a polycyclic heterocyclyl group wherein two rings of the polycyclic heterocyclyl group share two adjacent carbon atoms or heteroatoms.

[0123] In the present invention, "q" represents an average value. For example, when 1≤q≤8, it means that the average value is a value greater than or equal to 1 and less than or equal to 8.

[0124] "Antibody" or "antibody unit" includes within its scope any portion of an antibody that binds. This unit can bind, reactively associate or complex with a receptor, antigen, or other receptor unit possessed by a targeted cell population. An antibody can be any protein or protein-like molecule that can bind, complex or react with a portion of a cell population to be treated or bioengineered.

[0125] The antibodies of the present invention can specifically bind to antigens. Designed antigens include: tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth or differentiation (such as known or predicted functional), lymphokines, cytokines, factors involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis. Tumor-associated antigens can be cluster differentiation antigens (such as CD proteins). The antigens bound by the antibodies of the present invention can be one or a subset of the above classifications, and other subsets contain other molecules / antigens with special properties.

[0126] The antibodies used in the dual-drug linker assembly unit and dual-drug targeting linker-drug conjugate of the present invention include, but are not limited to, antibodies against tumor-associated antigens of cell surface receptors. Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below, including the names and gene bank accession numbers. Antibodies targeting corresponding tumor-associated antigens include all amino acid sequence variants and isotypes, with at least 70%, 80%, 85%, 90% or 95% homology with the sequences confirmed in the references, or have biological properties and characteristics that are completely consistent with the tumor-associated antigen sequences in the references.

[0127] Tumor-associated antigens include: BMPR1B (Genbank accession number: NM-001203), E16 (Genbank accession number: NM-003486), STEAP1 (Genbank accession number: NM-012449), 0772P (Genbank accession number: AF361486), MPF (Genbank accession number: NM-005823), Napi3b (Genbank accession number: NM-006424), Sema 5b (Genbank accession number: AB040878), PSCAhlg (Genbank accession number: AY358628), ETBR (Genbank accession number: AY275463), MSG783 (Genbank accession number: NM-017763), STEAP2 (Genbank accession number: AF455138), TrpM4 (Genbank accession number: NM-017636), CRIPTO (Genbank accession number: NP-003203 or NM-00 3212), CD21 (Genbank accession number: M26004), CD79B (Genbank accession number: NM-000626), FcRH2 (Genbank accession number: NM-030764), HER2 (Genbank accession number: M11730), NCA (Genbank accession number: M18728), MDP (Genbank accession number: BC017023), IL20Rα (Genbank accession number: AF184971), Brevican (Genbank accession number: AF184972), nbank accession number: AF229053), EpHB2R (Genbank accession number: NM-004442), GEDA (Genbank accession number: AY260763), BAFF-R (Genbank accession number: AF1164546), CD22 (Genbank accession number: AK026467), CD79a (Genbank accession number: NP-001774.1), CXCR5 (Genbank accession number: NP-001701.1), HLA-DOB ( (Genbank accession number: NP-002111.1), P2X5 (Genbank accession number: NP-002552.2), CD72 (Genbank accession number: NP-001773.1), LY64 (Genbank accession number: NP-005573.1), FcRH1 (Genbank accession number: NP-443170.1), IRTA2 (Genbank accession number: NP-112571.1), TENB2 (Genbank accession number: AF179274).

[0128] As used herein, "drug" or "D" refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugate of the present invention. Further, the drug includes a cytotoxic compound for cancer treatment, a protein or polypeptide with biological activity, including but not limited to camptothecin derivatives, such as SN-38, Dxd, Dx-8951, compounds acting on microtubules, such as Eribulin, MMAE, MMAF, maytansine, etc. (structure as follows).

[0129]

[0130]

[0131] According to the intracellular drug release mechanism, the "linkers" or "antibody drug conjugate linkers" described in this article can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0132] For dual-drug antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome to release the small molecule drug, the active small molecule composed of the linker and the amino acid residues of the antibody.

[0133] Cleavable linkers cleave within target cells and release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically labile linkers and enzyme-labile linkers.

[0134] Chemically labile linkers can selectively cleave due to differences in plasma and cytoplasmic properties, such as pH, glutathione concentration, etc. Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by intralysosomal proteases, such as cathepsins or plasmin. This peptide connection is considered to be very stable in plasma because proteases are usually inactive outside cells due to the unfavorable extracellular pH and serum protease inhibitors. In view of the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody drug conjugates.

[0135] "Self-stabilizing linker" refers to a linker structure that connects the anti-tumor compound to the antibody in a dual-drug antibody drug conjugate.

[0136] The present invention provides a dual-drug link assembly unit, which can be linked with a targeting linker to obtain a corresponding dual-drug targeting linker-drug conjugate, which can target tumor cells, reduce toxic side effects on normal cells, and effectively overcome drug resistance, thereby achieving a synergistic anti-tumor effect. Compared with the ADC Ds-8201 that has been on the market, the ADC provided by the present invention has significantly improved inhibitory effects on HER2-positive cell lines N87 and SK-BR-3d. The dual-drug link assembly unit and the dual-drug targeting linker-drug conjugate of the present invention have broad application prospects in the preparation of anti-tumor drugs for prevention and / or treatment.

[0137] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0138] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0139] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0140] Example 1. Preparation of the dual-drug linked assembly unit formula XIa of the present invention

[0141]

[0142] Step 1: Preparation of intermediate L-3

[0143]

[0144] The preparation method of compound L-2 refers to Chinese patent application No. 2020107518214.

[0145] Compound L-2 (179 mg, 0.15 mmol), compound L-1 (87 mg, 0.15 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (69 mg, 0.18 mmol), N,N-diisopropylethylamine (29 mg, 0.227 mmol), N,N-dimethylformamide (2 mL) were added to a 25 mL reaction bottle, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by TLC chromatography (developing solvent: dichloromethane: methanol = 10:1) to obtain intermediate L-3, weighing 132 mg, with a yield of 52.3%.

[0146] Step 2: Preparation of intermediate L-4

[0147]

[0148] Add intermediate L-3 (30 mg, 0.017 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (2.7 mg, 0.017 mmol) and N,N-dimethylformamide (1 mL) into a 25 mL reaction bottle and stir at room temperature for 30 minutes. The reaction is complete when monitored by TLC and used directly in the next step.

[0149] Step 3: Preparation of intermediate L-6

[0150]

[0151] In a 25 mL reaction bottle, intermediate L-4 (30 mg, 0.02 mmol), intermediate L-5 (26 mg, 0.07 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (25 mg, 0.07 mmol), N,N-diisopropylethylamine (9.2 mg, 0.07 mmol), N,N-dimethylformamide (3 mL) were added and reacted at room temperature for 30 minutes. TLC showed that the raw material L-4 disappeared and the reaction was complete. The mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by TLC chromatography (dichloromethane: methanol = 10:1) to obtain intermediate L-6 weighing 17 mg with a yield of 47%.

[0152] Step 4: Preparation of intermediate L-8

[0153]

[0154] In a 25 mL reaction bottle, add intermediate L-7 (60 mg, 0.071 mmol), L-1 (41 mg, 0.071 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (32.5 mg, 0.085 mmol), N,N-diisopropylethylamine (13.8 mg, 0.101 mmol), N,N-dimethylformamide (1 mL), stir at room temperature for 0.5 h, TLC monitoring shows that the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and the residue is purified by TLC chromatography (dichloromethane: methanol = 10:1) to obtain intermediate L-8, weighing 58.4 mg, with a yield of 60%.

[0155] Step 5: Preparation of intermediate L-9

[0156]

[0157] L-8 (10 mg, 0.007 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (2 mg, 0.010 mmol), and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction bottle and stirred at room temperature for 10 minutes. TLC showed that L-8 disappeared and the reaction was complete, and it was directly used in the next step.

[0158] Step 6: Preparation of Compound XIa

[0159]

[0160] In a 25 mL reaction bottle, add intermediate L-6 (10 mg, 0.005 mmol), L-9 (6 mg, 0.005 mmol), N,N-dimethylformamide (1.5 mL), N,N-diisopropylethylamine (1.2 mg, 0.007 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (2 mg, 0.006 mmol), cool in an ice-water bath and stir to react for 1 hour. TLC shows that L-6 disappears and the reaction is complete. Concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by TLC thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound XIa, weighing 11.7 mg. Yield 72%.

[0161] Example 2: Preparation of the dual-drug linker assembly unit XIb of the present invention

[0162]

[0163] Step 1: Preparation of compound XIb

[0164]

[0165] In a 25 mL reaction bottle, intermediate L-6 (10 mg, 0.005 mmol), intermediate L-7 (5.8 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (2.5 mg, 0.006 mmol), N,N-diisopropylethylamine (1.2 mg, 0.007 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIb, weighing 12 mg, with a yield of 96%.

[0166] Example 3. Preparation of the dual-drug linked assembly unit formula XIc of the present invention

[0167]

[0168] Step 1: Preparation of compound C-1

[0169]

[0170] Compound L-1 (100 mg, 0.18 mmol), compound C (200 mg, 0.18 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (101 mg, 0.27 mmol), N,N-diisopropylethylamine (45 mg, 0.35 mmol), N,N-dimethylformamide (4 mL) were added to a 25 mL reaction bottle. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by TLC chromatography (developing solvent: dichloromethane: methanol = 10:1) to obtain intermediate C-1, weighing 200 mg, with a yield of 67%.

[0171] Step 2: Preparation of compound C-2

[0172]

[0173] Add intermediate C-1 (30 mg, 0.020 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (4.5 mg, 0.030 mg), and N,N-dimethylformamide (1 mL) into a 25 mL reaction bottle and stir at room temperature for 30 minutes. The reaction is complete when monitored by TLC and used directly in the next step.

[0174] Step 3: Preparation of Compound XIc

[0175]

[0176] In a 25 mL reaction bottle, intermediate L-6 (10 mg, 0.006 mmol), intermediate C-2 (8.2 mg, 0.006 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (3.42 mg, 0.009 mmol), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIc, weighing 14 mg, with a yield of 67%.

[0177] Example 4. Preparation of the dual-drug linker assembly unit formula XId of the present invention

[0178]

[0179] Step 1: Preparation of compound XId

[0180]

[0181] In a 25 mL reaction bottle, intermediate L-6 (10 mg, 0.006 mmol), intermediate C (5 mg, 0.006 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.42 mg, 0.009 mmol), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound XId, weighing 8 mg, with a yield of 50%.

[0182] Example 5. Preparation of the dual-drug linked assembly unit formula XIe of the present invention

[0183]

[0184] Step 1: Preparation of compound XIe

[0185]

[0186] Add intermediate L-12 (15 mg, 0.008 mmol), L-9 (10 mg, 0.008 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.6 mg, 0.010 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, and the disappearance of L-12 indicates the completion of the reaction by LCMS monitoring. Direct reverse phase preparation yields 12 mg of a yellow solid with a yield of 52%.

[0187] Example 6: Preparation of the dual-drug linker assembly unit XIf of the present invention

[0188]

[0189] Step 1: Preparation of intermediate L-11

[0190]

[0191] L-10 (460 mg, 1.475 mmol), 3-aminopentanedioic acid (200 mg, 1.359 mmol), sodium bicarbonate (148 mg, 1.767 mmol), 6 mL of tetrahydrofuran, and 12 mL of water were added to a 25 mL reaction bottle. The mixture was stirred at room temperature for 24 hours, filtered, and the filtrate was extracted with 50 mL of ethyl acetate. The aqueous phase was separated and adjusted to pH 2 with hydrochloric acid, and extracted with 100 mL of ethyl acetate: methanol = 40:1. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain compound L-11, weighing 35 mg, with a yield of 7%.

[0192] 1 H NMR(400MHz, CDCl3)δ12.01(s,1H),11.90(s,1H),8.14(s,1H),δ7.83(s,1H),7.74(s,1H),4.24(m,1H) ,4.00(t,J=9.1Hz,2H),2.42(dd,J=12.8Hz,2H),2.15(t,J=9.8Hz,2H),1.63(m,2H),1.38-1.20(m,4H).

[0193] Step 2: Preparation of intermediate L-12

[0194]

[0195] L-11 (16 mg, 0.047 mmol), L-4 (20 mg, 0.014 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)uronium hexafluorophosphate (18 mg, 0.047 mmol), N,N-diisopropylethylamine (6.1 mg, 0.047 mmol) were added to a 25 mL reaction bottle and stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10:1) to obtain intermediate L-12, weighing 10 mg, with a yield of 40%.

[0196] Step 3: Preparation of Compound XIf

[0197]

[0198] Compound L-12 (10 mg, 0.007 mmol), L-7 (5.8 mg, 0.007 mmol), N,N-dimethylformamide (1.5 mL), N,N-diisopropylethylamine (1.8 mg, 0.014 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate (2.5 mg, 0.010 mmol) were added to a 25 mL reaction bottle and stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 8:1) to obtain compound XIf, weighing 6.8 mg.

[0199] Example 7. Preparation of the dual-drug linked assembly unit formula XIg of the present invention

[0200]

[0201] Step 1: Preparation of compound XIg

[0202]

[0203] Add intermediate L-12 (15 mg, 0.008 mmol), C-2 (12 mg, 0.008 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.6 mg, 0.010 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, and the disappearance of L-12 indicates the completion of the reaction by LCMS monitoring. Direct reverse phase preparation yields 13 mg of a white solid with a yield of 50%.

[0204] Example 8. Preparation of the dual-drug linker assembly unit XIh of the present invention

[0205]

[0206] Step 1: Preparation of compound XIg

[0207]

[0208] Add intermediate L-12 (15 mg, 0.008 mmol), C (9 mg, 0.008 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.6 mg, 0.010 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, and monitor the disappearance of L-12 by LCMS, indicating the completion of the reaction. Direct reverse phase preparation gave 10 mg of a white solid with a yield of 43%.

[0209] Example 9. Preparation of the dual-drug linked assembly unit formula XIi of the present invention

[0210]

[0211] Step 1: Preparation of compound E-1

[0212]

[0213] In a 25 mL reaction bottle, add intermediate E (100 mg, 0.19 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (109 mg, 0.29 mmol), N,N-diisopropylethylamine (49 mg, 0.38 mmol), N,N-dimethylformamide (4 mL), stir at room temperature for 5 minutes, slowly add intermediate L-5 (34 mg, 0.19 mmol) and react at room temperature for 30 minutes. TLC shows that the reaction is complete, and the mixture is concentrated under reduced pressure to remove N,N-dimethylformamide. The residue is purified by thin layer chromatography (dichloromethane: methanol = 6:1) to obtain compound E-1, weighing 34 mg, with a yield of 26%.

[0214] 1 H NMR (400MHz, CDCl3) δ12.74(s,1H),12.56(s,1H),10.10(s,1H),8.51(d,J=2.3Hz,1H),8.42(d,J=2.3Hz,1H) ,8.16(d,J=1.2Hz,1H), δ7.83(s,1H),7.74(s,1H),3.72(m,4H),3.50-2.50(m,30H),2.22(dd,J=12.8Hz,2H).

[0215] Step 2: Preparation of compound E-2

[0216]

[0217] In a 25 mL reaction bottle, add intermediate E-1 (50 mg, 0.70 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (54 mg, 0.14 mmol), N,N-diisopropylethylamine (18 mg, 0.14 mmol), N,N-dimethylformamide (4 mL), stir at room temperature for 5 minutes, slowly add intermediate L-4 (58 mg, 0.04 mmol) in N,N-dimethylformamide (1 mL) solution and react at room temperature for 30 minutes. TLC shows that the reaction is complete, and the mixture is concentrated under reduced pressure to remove N,N-dimethylformamide. The residue is purified by thin layer chromatography (dichloromethane: methanol = 8:1) to obtain compound E-2, weighing 32 mg, with a yield of 38%.

[0218] Step 3: Preparation of Compound XIi

[0219]

[0220] In a 25 mL reaction bottle, intermediate E-2 (10 mg, 0.005 mmol), intermediate L-7 (3.9 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (2.8 mg, 0.007 mmol), N,N-diisopropylethylamine (1.2 mg, 0.010 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound XIi, weighing 8 mg, with a yield of 57%.

[0221] Example 10. Preparation of the dual-drug linker assembly unit formula XIj of the present invention

[0222]

[0223] Step 1: Preparation of Compound XIj

[0224]

[0225] In a 25 mL reaction bottle, intermediate E-2 (10 mg, 0.005 mmol), intermediate C (5.3 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (2.8 mg, 0.007 mmol), N,N-diisopropylethylamine (1.2 mg, 0.010 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound XIj, weighing 7.8 mg, with a yield of 56%.

[0226] Example 11. Preparation of the dual-drug linker assembly unit formula XIk of the present invention

[0227]

[0228] Step 1: Preparation of compound B-1

[0229]

[0230] Compound L-7 (100 mg, 0.12 mmol), compound M (143 mg, 0.12 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (68 mg, 0.18 mmol), N,N-diisopropylethylamine (30 mg, 0.24 mmol), N,N-dimethylformamide (4 mL) were added to a 25 mL reaction bottle and stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by TLC chromatography (developing solvent: dichloromethane: methanol = 10:1) to obtain intermediate C-1, weighing 80 mg, with a yield of 34%.

[0231] Step 2: Preparation of compound B-2

[0232]

[0233] Add intermediate B-2 (80 mg, 0.04 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (9.1 mg, 0.06 mg), and N,N-dimethylformamide (1 mL) into a 25 mL reaction bottle and stir at room temperature for 30 minutes. The reaction is complete when monitored by TLC and used directly in the next step.

[0234] Step 3: Preparation of Compound XIk

[0235]

[0236] In a 25 mL reaction bottle, intermediate E-2 (10 mg, 0.005 mmol), intermediate B-2 (10 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (2.8 mg, 0.007 mmol), N,N-diisopropylethylamine (1.2 mg, 0.010 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIk, weighing 13 mg, with a yield of 65%.

[0237] Example 12: Preparation of the dual-drug linker assembly unit formula XI1 of the present invention

[0238]

[0239] Step 1: Preparation of compound C-3

[0240]

[0241] In a 25 mL reaction bottle, add intermediate E-1 (50 mg, 0.70 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (54 mg, 0.14 mmol), N,N-diisopropylethylamine (18 mg, 0.14 mmol), N,N-dimethylformamide (4 mL), stir at room temperature for 5 minutes, slowly add intermediate C-2 (58 mg, 0.04 mmol) in N,N-dimethylformamide (1 mL) solution and react at room temperature for 30 minutes. TLC shows that the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by thin layer chromatography (dichloromethane: methanol = 8:1) to obtain compound C-3, weighing 33 mg, with a yield of 38%.

[0242] Step 2: Preparation of Compound XI1

[0243]

[0244] In a 25 mL reaction bottle, intermediate C-3 (10 mg, 0.005 mmol), intermediate L-7 (3.9 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (2.8 mg, 0.007 mmol), N,N-diisopropylethylamine (1.2 mg, 0.010 mmol), N,N-dimethylformamide (1 mL) were added, and the reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIl, weighing 7.3 mg, with a yield of 55%.

[0245] Example 13. Preparation of the dual-drug linker assembly unit formula XIm of the present invention

[0246]

[0247] Step 1: Preparation of compound XIm

[0248]

[0249] In a 25 mL reaction bottle, intermediate L-4 (15 mg, 0.010 mmol), intermediate L-5 (1 mg, 0.005 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (3.8 mg, 0.010 mmol), N,N-diisopropylethylamine (2.0 mg, 0.015 mmol), N,N-dimethylformamide (3 mL) were added and reacted at room temperature for 30 minutes. TLC showed that the raw material L-4 disappeared and the reaction was complete. The mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by TLC chromatography (dichloromethane: methanol = 10:1) to obtain product XIm weighing 5 mg with a yield of 31%.

[0250] Example 14: Preparation of the dual-drug linked assembly unit XIn of the present invention

[0251]

[0252] Step 1: Preparation of compound XIn

[0253]

[0254] Add intermediate L-11 (2 mg, 0.004 mmol), L-4 (12 mg, 0.008 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.6 mg, 0.010 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, and monitor the disappearance of L-12 by LCMS, indicating that the reaction is complete. Direct reverse phase preparation gave 8 mg of a white solid with a yield of 61%.

[0255] Example 15. Preparation of the dual-drug linked assembly unit formula XIo of the present invention

[0256]

[0257] Step 1: Preparation of compound XIo

[0258]

[0259] Add intermediate L-11 (2 mg, 0.004 mmol), L-9 (9.5 mg, 0.008 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (1.5 mg, 0.012 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (3.6 mg, 0.010 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, and the disappearance of L-12 indicates the completion of the reaction by LCMS monitoring. Direct reverse phase preparation yields 6 mg of a yellow solid with a yield of 56%.

[0260] Example 16. Preparation of the dual-drug linker assembly unit formula XIp of the present invention

[0261]

[0262] Step 1: Preparation of intermediate D-1

[0263]

[0264] Add intermediate D-0 (50 mg, 0.077 mmol), E (59 mg, 0.081 mmol), N,N-dimethylformamide (4 mL), N,N-diisopropylethylamine (1.2 mg, 0.115 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (35 mg, 0.092 mmol) to a 25 mL reaction bottle, stir at room temperature for 1 hour, TLC shows that D-0 disappears, and the reaction is complete. Concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by TLC thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound D-1, weighing 65 mg. Yield 62%.

[0265] Step 2: Preparation of intermediate D-2

[0266]

[0267] D-1 (65 mg, 0.048 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (11 mg, 0.072 mmol), N,N-dimethylformamide (2 mL) were added to a 10 mL reaction bottle, and the mixture was stirred at room temperature for 10 minutes. TLC showed that D-1 disappeared and the reaction was complete. 35 mg of white solid was obtained by reverse phase preparation. The yield was 65%.

[0268] Step 3: Preparation of intermediate D-3

[0269]

[0270] D-3-2 (100 mg, 0.180 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (42 mg, 0.270 mmol), N,N-dimethylformamide (2 mL) were added to a 10 mL reaction bottle and stirred at room temperature for 10 minutes. TLC showed that D-3-2 disappeared and the reaction was complete. The reaction solution was added dropwise to a flask containing D-3-1 (32 mg, 0.150 mmol), N,N,N′,N′-tetramethyl-O-(7-nitrogen)-1,4-dimethylformamide (2 mL). Heterobenzotriazole-1-yl) urea hexafluorophosphate (85 mg, 0.225 mmol), N,N-diisopropylethylamine (30 mg, 0.300 mmol), N,N-dimethylformamide (2 mL) were placed in a reaction bottle and stirred at room temperature for 30 minutes. LCMS monitored that the reaction of the raw materials was complete. Water (5 mL) was added, and the mixture was extracted with ethyl acetate (15 mL×3). The mixture was dried over anhydrous sodium sulfate, filtered, and dried by spin drying. A small amount of methanol was added and reverse phase preparation was performed to obtain 50 mg of an oily liquid with a yield of 63%.

[0271] 1H NMR (400MHz, CDCl3) δ7.86(s,1H),7.81(s,1H),4.09(m,2H),3.70-3.50(m,12H),2.42(m,4H),2.11-1.62(m,8H),1.37(s,18H).

[0272] Step 4: Preparation of intermediate D-4

[0273]

[0274] D-3 (50 mg, 0.095 mmol) was added to a 10 mL reaction bottle, trifluoroacetic acid (1 mL), and dichloromethane (1 mL). The reaction was stirred at room temperature for 4 h. LCMS monitored that the reaction of the raw material was complete. The dichloromethane was spun off, and a small amount of methanol was added. The mixture was prepared by reverse phase to obtain 30 mg of an oily liquid with a yield of 77%.

[0275] 1 H NMR (400MHz, CDCl3)12.09(s,1H),11.91(s,1H),δ7.86(s,1H),7.81(s,1H),4.12(m,2H),3.65-3.47(m,12H),2.45(m,4H),2.08-1.67(m,8H).

[0276] Step 5: Preparation of intermediate D-5

[0277]

[0278] Add intermediate D-4 (30 mg, 0.072 mmol), L-2 (27 mg, 0.024 mmol), N,N-dimethylformamide (3 mL), N,N-diisopropylethylamine (9 mg, 0.072 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (27 mg, 0.072 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, TLC shows that L-2 disappears, and the reaction is complete. Concentrate under reduced pressure to remove N,N-dimethylformamide, add a small amount of methanol to the residue, and prepare by reverse phase to obtain 23 mg of white solid, with a yield of 64%.

[0279] Step 6: Preparation of Compound XIp

[0280]

[0281] Add intermediate D-5 (23 mg, 0.015 mmol), D-2 (17 mg, 0.015 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (3 mg, 0.022 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (8 mg, 0.022 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 minutes, TLC shows that D-5 disappears, and the reaction is complete. Direct reverse phase preparation gives 15 mg of white solid, with a yield of 38%.

[0282] Example 17: Preparation of intermediate GGFGE

[0283]

[0284] Step 1: Preparation of compound D2

[0285]

[0286] In a 500mL reaction bottle, intermediate D1 (20g, 56.49mmol) and acetonitrile (200mL) were added. After complete clarification, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11g, 56.49mmol) and N-hydroxysuccinimide (7g, 56.49mmol) were added. The reaction was stirred at room temperature for 12h. TLC showed that the reaction was complete. The product was filtered and the solid was dried to obtain compound D2, weighing 22g, with a yield of 90%.

[0287] Step 2: Preparation of compound D3

[0288]

[0289] In a 500mL reaction bottle, add the intermediate L-phenylalanine (8g, 48.48mmol), sodium bicarbonate (8g, 96.96mmol), water (200mL), and after complete clarification, dissolve compound D2 (22g, 48.48mmol) in ethylene glycol dimethyl ether (50mL) and slowly add it to the above reaction solution. Stir the reaction at room temperature for 12h. TLC shows that the reaction is complete. Concentrate under reduced pressure to remove ethylene glycol dimethyl ether. The residue is dropped into 0.5M hydrochloric acid aqueous solution (500mL). A large amount of solid is precipitated. Filter and dry the solid to obtain compound D3, weighing 15g, with a yield of 62%.

[0290] 1H NMR (400MHz, CDCl3)12.51(s,1H),9.04(s,1H),8.31(s,1H),7.95(s,1H),7.90(d,J=8.0Hz,2H),7.56(d,J=7.8Hz,2H),7.38-7.28(m,4H),7.19- 7.14(m,5H),4.85(t,J=8.2Hz,1H),4.71(d,J=8.2Hz,2H),4.39(t,J=8.4 Hz,1H),4.10-3.83(m,4H),3.12(d,J=9.6Hz,1H),2.85(d,J=9.6Hz,1H).

[0291] Step 3: Preparation of compound D4

[0292]

[0293] In a 500 mL reaction bottle, add intermediate D3 (15 g, 29.9 mmol), acetonitrile (200 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 g, 29.9 mmol), N-hydroxysuccinimide (4 g, 29.9 mmol), stir at room temperature for 12 h, TLC shows the reaction is complete, filter, and dry the solid to obtain compound D4, weighing 13 g, with a yield of 72%.

[0294] Step 4: Preparation of compound D5

[0295]

[0296] In a 500mL reaction bottle, add the intermediate glycine (3g, 40.0mmol), sodium bicarbonate (7g, 80.0mmol), and water (150mL). After complete clarification, dissolve compound D4 (13g, 40.0mmol) in ethylene glycol dimethyl ether (40mL) and slowly add it to the above reaction solution. Stir the reaction at room temperature for 12h. TLC shows that the reaction is complete. Concentrate under reduced pressure to remove the solvent. The residue is dropped into 0.5M hydrochloric acid aqueous solution (300mL). A large amount of solid is precipitated. Filter and dry the solid to obtain compound D5, weighing 15g, with a yield of 71%.

[0297] 1H NMR (400MHz, CDCl3)13.01(s,1H),9.01(s,1H),8.27(s,1H),7.98(s,1H),7.89(d,J=8.0Hz,2H),7.54(d,J=7.8Hz,2H),7.34-7.23(m,4H),7.19- 7.14(m,5H),4.75(t,J=8.2Hz,1H),4.61(d,J=8.2Hz,2H),4.30(t,J=8.4 Hz,1H),4.04-3.83(m,6H),3.10(d,J=9.6Hz,1H),2.75(d,J=9.6Hz,1H).

[0298] Step 5: Preparation of compound D6

[0299]

[0300] In a 25 mL reaction bottle, intermediate D5 (200 mg, 0.36 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (163 mg, 0.43 mmol), N,N-diisopropylethylamine (66.9 mg, 0.54 mmol), N,N-dimethylformamide (5 mL) were added. After stirring at room temperature for 5 min, eribulin (263.1 mg, 0.36 mmol) was added. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound D6, weighing 300 mg, with a yield of 50%.

[0301] Step 6: Preparation of compound GGFGE

[0302]

[0303] In a 25 mL reaction bottle, intermediate D6 (20 mg, 0.016 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (5 mg, 0.032 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane:methanol=10:1) to obtain compound GGFGE, weighing 10 mg, with a yield of 62%.

[0304] Example 18. Preparation of the dual-drug linker assembly unit formula XIq of the present invention

[0305]

[0306] Step 1: Preparation of compound M-1

[0307]

[0308] Compound M (1.00 g, 4.9 mmol) and solvent N,N-dimethylformamide (15 mL) were added to a 25 mL reaction bottle in sequence. After the reaction solution was clarified, potassium carbonate (2.43 g, 17.6 mmol), potassium iodide (1.63 g, 9.8 mmol), and 2-chloroethoxy-2-ethoxydiethanol (1.63 g, 9.8 mmol) were added in sequence. The reaction was stirred at 110 ° C for 12 h. TLC showed that the reaction was complete. After filtration, the mixture was concentrated under reduced pressure to remove excess N,N-dimethylformamide. The residue was purified by HPLC to obtain intermediate M-1, weighing 1.5 g, with a yield of 83%.

[0309] 1 H NMR (400MHz, CDCl3) δ7.32-7.29(m,5H),4.51(s,2H),4.43(s,1H),3.72–3.69(m,4H),3.55–3.50(m,8H),2.65–2.50(m,6H),1.59–1.34(m,8H).

[0310] Step 2: Preparation of compound M-2

[0311]

[0312] Add intermediate M-1 (1.50 g, 3.95 mmol), tetrabutylammonium bromide (2.10 g, 1.58 mmol), and dichloromethane (10 mL) to a 25 mL reaction bottle. Stir and react at 0°C for 30 min. Then, dissolve sodium hydroxide (1.26 g, 31.60 mmol) in water (1.2 mL). After clarification, slowly add it dropwise to the above reaction solution. Continue to react at 0°C for 10 min, then remove from the ice bath and react at room temperature for 3 h. LC-MS monitors the reaction to be complete. Adjust the reaction solution to weak acidity with dilute hydrochloric acid at pH = 3, extract dichloromethane (10 mL * 3), combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and remove excess dichloromethane by vacuum distillation to obtain 1.7 g of the target product with a yield of 75%, which is directly used in the next step.

[0313] Step 3: Preparation of Compound M-3

[0314]

[0315] In a 25 mL reaction bottle, intermediate M-2 (1 g, 1.57 mmol), methanol (10 mL), and a catalytic amount of palladium carbon were added in sequence. The mixture was protected by hydrogen and stirred at room temperature for 2 h. LC-MS showed that the reaction was complete. The palladium carbon was removed by filtration, and the excess solvent was concentrated under reduced pressure. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-3, weighing 900 mg, with a yield of 75%.

[0316] 1 H NMR (400MHz, CDCl3) δ3.72–3.69(m,4H),3.55–3.50(m,12H),2.65–2.42(m,10H),1.59–1.34(m,8H),1.49(s,18H).

[0317] Step 4: Preparation of Compound M-4

[0318]

[0319] In a 25mL reaction bottle, add intermediate M-3 (900mg, 1.57mmol), THF (10mL), water (2.5mL), and after the reaction solution is clarified, add sodium bicarbonate (210mg, 2.50mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (530mg, 1.57mmol). Stir the reaction at room temperature for 2h. LC-MS shows that the reaction is complete. Then, the excess solvent is concentrated under reduced pressure, and the residue is purified by thin layer chromatography (dichloromethane: methanol = 10:1) to obtain compound M-4, weighing 1g, with a yield of 74%.

[0320] 1 H NMR (400MHz, CDCl3) δ7.90(d,J=8.9Hz,2H),7.55(d,J=9.2Hz,2H),7.38-7.28(m,4H),4.70(d,J=11.2Hz,2H),4.4 6(t,J=11.2Hz,1H),3.71–3.67(m,4H),3.53–3.48(m,12H),2.55–2.41(m,10H),1.51–1.24(m,8H),1.41(s,18H).

[0321] Step 5: Preparation of Compound M-5

[0322]

[0323] In a 25 mL reaction bottle, add intermediate M-4 (1 g, 1.54 mmol), dichloromethane (5 mL), and trifluoroacetic acid (5 mL) in sequence, and stir at room temperature for 2 h. LC-MS shows that the reaction is complete. Concentrate under reduced pressure to remove excess solvent, and purify the residue by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-5, weighing 600 mg, with a yield of 75%.

[0324] Step 6: Preparation of Compound M-6

[0325]

[0326] In a 25 mL reaction bottle, add intermediate GGFGE (50 mg, 0.05 mmol), intermediate M-5 (50 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (38 mg, 0.10 mmol), N,N-diisopropylethylamine (12.9 mg, 0.10 mmol), N,N-dimethylformamide (4 mL), stir at room temperature for 30 min, TLC shows that the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-6, weighing 50 mg, with a yield of 33%.

[0327] Step 7: Preparation of Compound M-7

[0328]

[0329] In a 25 mL reaction bottle, intermediate M-6 (30 mg, 0.02 mmol), intermediate L-7 (16 mg, 0.02 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (7.6 mg, 0.02 mmol), N,N-diisopropylethylamine (2.58 mg, 0.02 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-7, weighing 20 mg, with a yield of 44%.

[0330] Step 8: Preparation of Compound M-8

[0331]

[0332] In a 25 mL reaction bottle, add intermediate M-7 (20 mg, 0.008 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (2.45 mg, 0.016 mmol), N,N-dimethylformamide (3 mL), stir at room temperature for 30 min, TLC shows the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and the residue is purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-8, weighing 15 mg, with a yield of 75%.

[0333] Step 9: Preparation of Compound XIq

[0334]

[0335] In a 25 mL reaction bottle, intermediate M-8 (10 mg, 0.004 mmol), intermediate Mal-5PEG-acid (1.60 mg, 0.004 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (1.52 mg, 0.004 mmol), N,N-diisopropylethylamine (0.52 mg, 0.004 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIq, weighing 7 mg, with a yield of 44%.

[0336] Example 19. Preparation of the dual-drug linked assembly unit formula XIr of the present invention

[0337]

[0338] Step 1: Preparation of compound XIr

[0339]

[0340] In a 25 mL reaction bottle, intermediate M-8 (10 mg, 0.004 mmol), intermediate Mal-8PEG-acid (1.70 mg, 0.004 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (1.52 mg, 0.004 mmol), N,N-diisopropylethylamine (0.52 mg, 0.004 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIr, weighing 6.5 mg, with a yield of 45%.

[0341] Example 20. Preparation of the dual-drug linked assembly unit formula XIs of the present invention

[0342]

[0343] Step 1: Preparation of compound M-9

[0344]

[0345] In a 25 mL reaction bottle, add intermediate GGFGE (50 mg, 0.05 mmol), intermediate M-5 (13 mg, 0.03 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (38 mg, 0.10 mmol), N,N-diisopropylethylamine (13 mg, 0.10 mmol), N,N-dimethylformamide (4 mL), stir at room temperature for 30 min, TLC shows that the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-9, weighing 40 mg, with a yield of 81%.

[0346] Step 2: Preparation of compound M-10

[0347]

[0348] In a 25 mL reaction bottle, add intermediate M-9 (20 mg, 0.007 mmol), 1,8-diazobispiro[5.4.0]undec-7-ene (2.35 mg, 0.014 mmol), N,N-dimethylformamide (3 mL), stir at room temperature for 30 min, TLC shows the reaction is complete, concentrate under reduced pressure to remove N,N-dimethylformamide, and the residue is purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound M-10, weighing 15 mg, with a yield of 83%.

[0349] Step 3: Preparation of Compound XIs

[0350]

[0351] In a 25 mL reaction bottle, intermediate M-10 (10 mg, 0.004 mmol), intermediate Mal-8PEG-acid (2 mg, 0.004 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (2 mg, 0.004 mmol), N,N-diisopropylethylamine (1 mg, 0.004 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the mixture was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIs, weighing 6.5 mg, with a yield of 45%.

[0352] Example 21. Preparation of the dual-drug linked assembly unit formula XIt of the present invention

[0353]

[0354] Step 1: Preparation of compound XIt

[0355]

[0356] In a 25 mL reaction bottle, intermediate M-8 (10 mg, 0.004 mmol), intermediate Mal-2PEG-acid (1.34 mg, 0.004 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (1.52 mg, 0.004 mmol), N,N-diisopropylethylamine (0.52 mg, 0.004 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIt, weighing 5.9 mg, with a yield of 53%.

[0357] Example 22. Preparation of the dual-drug linker assembly unit formula XIu of the present invention

[0358]

[0359] Step 1: Preparation of compound XIu

[0360]

[0361] In a 25 mL reaction bottle, intermediate M-8 (10 mg, 0.004 mmol), intermediate L-2 (1.34 mg, 0.004 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (1.52 mg, 0.004 mmol), N,N-diisopropylethylamine (0.52 mg, 0.004 mmol), N,N-dimethylformamide (3 mL) were added, and the reaction was stirred at room temperature for 30 min. TLC showed that the reaction was complete, and the N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin layer chromatography (dichloromethane: methanol = 10: 1) to obtain compound XIu, weighing 5.6 mg, with a yield of 52%.

[0362] Example 23. Preparation of the dual-drug linked assembly unit formula XIv of the present invention

[0363]

[0364] Step 1: Preparation of intermediate N-1

[0365]

[0366] Add intermediate N-0 (2.0 g, 5.63 mmol) and N,N-dimethylformamide (10 mL) to a 25 mL reaction bottle, cool in an ice-water bath under nitrogen protection for 15 minutes, add sodium hydrogen (0.4 g, 8.44 mmol) in batches, stir for 5 minutes, then add tert-butyl bromoacetate (1.6 g, 8.44 mmol), slowly warm to room temperature, stir and react for 1 hour, TLC shows that N-0 disappears and the reaction is complete. Add water (15 mL), extract with ethyl acetate (30 mL*3), dry over anhydrous sodium sulfate, filter, spin dry, add a small amount of methanol, and prepare in reverse phase to obtain 1.7 g of oily liquid with a yield of 50%.

[0367] 1 H NMR (400MHz, CDCl3) δ7.55-7.33(m,10H),5.34(s,2H),5.02(s,2H),4.33(s,2 H), 4.28 (t, J = 11.2Hz, 1H), 3.71–3.46 (m, 3H), 2.40–2.25 (m, 2H), 1.42 (s, 9H).

[0368] Step 2: Preparation of intermediate N-2

[0369]

[0370] To a 25 mL reaction bottle, add N-1 (1.7 g, 3.62 mmol), palladium carbon (0.2 g, 10%), and methanol (20 mL). Under a hydrogen atmosphere, stir at room temperature for 2 hours. LCMS monitoring showed that N-1 disappeared, indicating that the reaction was complete. The mixture was filtered and dried to give 800 mg of a crude product, which was directly used for the next step.

[0371] Step 3: Preparation of intermediate N-3

[0372]

[0373] L-2 (413 mg, 1.96 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (744 mg, 1.96 mmol), DIEA (315 mg, 2.45 mmol), and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction bottle, and the reaction was stirred at room temperature for 10 min. N-2 (400 mg, 1.63 mmol) was then added dropwise, and stirring was continued for 30 minutes. LCMS monitored that the raw material reaction was complete, water (15 mL) was added, and the mixture was extracted with ethyl acetate (30 mL*3), dried over anhydrous sodium sulfate, filtered, and spin-dried. A small amount of methanol was added and reversed to obtain 500 mg of a white solid with a yield of 70%.

[0374] 1 H NMR (400MHz, CDCl3) δ12.22(s,1H),7.90(s,1H),7.85(s,1H),4.33(m,3H),4.02(t,J=11.2Hz,2H), 3.71–3.46(m,3H),2.46(m,1H),2.21(m,1H),2.02(t,J=9.8Hz,2H),1.61–1.24(m,6H),1.40(s,9H).

[0375] Step 4: Preparation of intermediate N-4

[0376]

[0377] N-3 (500 mg, 1.14 mmol) was added to a 10 mL reaction bottle. Trifluoroacetic acid (5 mL) and dichloromethane (5 mL) were added. The reaction was stirred at room temperature for 4 h. LCMS monitored that the reaction of the raw material was complete. The excess solvent was removed by rotary evaporation under reduced pressure. A small amount of methanol was added and 300 mg of oily liquid was obtained by reverse phase preparation. The yield was 78%. 1H NMR (400MHz, CDCl3) δ12.80(s,1H),12.25(s,1H),7.89(s,1H),7.80(s,1H),4.31(m,3H),4.00(t,J=11.2Hz,2 H),3.77–3.46(m,3H),2.45(m,1H),2.20(m,1H),2.05(t,J=9.8Hz,2H),1.63(m,2H),1.53(m,2H),1.32(m,2H).

[0378] Step 5: Preparation of intermediate N-5

[0379]

[0380] Add intermediate N-4 (40 mg, 0.105 mmol), N,N-dimethylformamide (3 mL), N,N-diisopropylethylamine (13 mg, 0.105 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (40 mg, 0.105 mmol) to a 25 mL reaction bottle, stir at room temperature for 10 min, then add GGFGE (27 mg, 0.026 mmol dissolved in 1 mL N,N-dimethylformamide) dropwise, continue stirring for 30 min, LCMS monitoring GGFGE disappears, the reaction is complete. Direct reverse phase preparation to obtain 20 mg of white solid, yield 54%.

[0381] Step 6: Preparation of Compound XIv

[0382]

[0383] Add intermediate N-5 (20 mg, 0.014 mmol), B (12 mg, 0.014 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (3 mg, 0.021 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (6 mg, 0.017 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 min, monitor N-5 disappearance by LCMS, the reaction is complete. Direct reverse phase preparation to obtain 15 mg of yellow solid, yield 48%.

[0384] Example 24. Preparation of the dual-drug linker assembly unit formula XIw of the present invention

[0385]

[0386] Step 1: Preparation of compound N-7

[0387]

[0388] Add intermediate N-6 (1.0 g, 3.25 mmol), N-6-0 (0.6 g, 3.89 mmol), sodium bicarbonate (1.1 g, 13.00 mmol), ethylene glycol dimethyl ether (15 mL), water (15 mL) to a 25 mL reaction bottle, stir at room temperature for 20 hours, and LCMS monitoring shows that most of the molecular weight is the target compound. After concentrating half of the solvent, directly reverse phase preparation gave 110 mg of colorless liquid with a yield of 10%.

[0389] 1 H NMR (400MHz, CDCl3) δ12.66(s,1H),12.01(s,1H),7.90(s,1H),7.76(s,1H),4.55(t,J=9.8Hz,1 H),4.05(t,J=11.2Hz,2H),2.33(t,J=9.2Hz,2H),2.05(m,4H),1.65(m,2H),1.32–1.28(m,4H).

[0390] Step 2: Preparation of Compound N-8

[0391]

[0392] Add intermediate N-7 (26 mg, 0.076 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (10 mg, 0.076 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (29 mg, 0.076 mmol) to a 25 mL reaction bottle, stir at room temperature for 10 min, then add GGFGE (20 mg, 0.019 mmol dissolved in 1 mL N,N-dimethylformamide) dropwise, continue stirring for 30 min, LCMS monitoring GGFGE disappears, the reaction is complete. Direct reverse phase preparation to obtain 18 mg of white solid, yield 69%.

[0393] Step 3: Preparation of Compound XIw

[0394]

[0395] Add intermediate N-8 (18 mg, 0.013 mmol), B-1 (6 mg, 0.013 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (3 mg, 0.020 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (6 mg, 0.015 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 min, monitor N-8 disappearance, reaction complete. Direct reverse phase preparation to obtain 14 mg of yellow solid, yield 60%.

[0396] Example 25. Preparation of the dual-drug linked assembly unit formula XIx of the present invention

[0397]

[0398] Step 1: Preparation of compound N-10

[0399]

[0400] Add intermediate N-10 (200 mg, 0.37 mmol), palladium carbon (20 mg, 10%), and methanol (5 mL) to a 25 mL reaction bottle, and stir at room temperature under a hydrogen atmosphere for 2 hours. LCMS monitoring shows that N-10 disappears, indicating that the reaction is complete. Directly filter and concentrate to obtain 150 mg of crude oily liquid with a yield of 90%.

[0401] Step 2: Preparation of Compound N-11

[0402]

[0403] Add intermediate N-10 (150 mg, 0.33 mmol), SM-1 (79 mg, 0.33 mmol), N,N-dimethylformamide (5 mL), N,N-diisopropylethylamine (65 mg, 0.50 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (152 mg, 0.40 mmol) to a 25 mL reaction bottle, stir at room temperature for 10 min, LCMS monitoring showed that most of the molecular weight was the target compound, and the reaction was complete. Direct reverse phase preparation gave 110 mg of oily liquid with a yield of 49%.

[0404] 1H NMR(400MHz, CDCl3)δ7.76(s,1H),7.55(s,1H),3.73–3.67(m,8H),3.52–3.48(m,8H),3.37(m,4H),3 .40(d,J=8.2Hz,2H),2.42–2.38(m,5H),2.06(m,1H),1.73-1.70(m,4H),1.42(s,18H),1.38(m,4H).

[0405] Step 3: Preparation of Compound N-12

[0406]

[0407] N-11 (110 mg, 0.16 mmol) was added to a 10 mL reaction bottle, trifluoroacetic acid (3 mL) and dichloromethane (3 mL). The mixture was stirred at room temperature for 4 h. LCMS monitored the complete reaction of the raw material. The excess solvent was removed by rotary evaporation under reduced pressure. A small amount of methanol was added and 60 mg of an oily liquid was obtained by reverse phase preparation. The yield was 67%. 1 H NMR (400MHz, CDCl3) δ12.10(s,1H),11.93(s,1H),7.86(s,1H),7.65(s,1H),3.61–3.52(m ,16H),3.40–3.37(m,6H),2.40–2.38(m,5H),2.03(m,1H),1.72-1.70(m,4H),1.36(m,4H).

[0408] Step 4: Preparation of Compound XIx

[0409]

[0410] Add intermediate N-12 (5 mg, 0.008 mmol), B (15 mg, 0.018 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (3 mg, 0.024 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (7 mg, 0.018 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 min, monitor N-12 disappearance, reaction complete. Direct reverse phase preparation to obtain 10 mg of yellow solid, yield 55%.

[0411] Example 26. Preparation of the dual-drug linker assembly unit formula XIy of the present invention

[0412]

[0413] Step 1: Preparation of Compound N-13

[0414]

[0415] Add intermediate L-2 (97 mg, 0.46 mmol), SM-1 (200 mg, 0.46 mmol), N,N-dimethylformamide (5 mL), N,N-diisopropylethylamine (89 mg, 0.68 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (209 mg, 0.55 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 min, LCMS monitoring L-2 disappears, the reaction is complete. Direct reverse phase preparation to obtain 200 mg of white solid, yield 69%.

[0416] 1 H NMR (400MHz, CDCl3) δ7.86 (s, 1H), 7.75 (s, 1H), 7.65-7.56 (m, 4H), 7.51-7.45 (m, 4H), 4.00 (t, J = 11.2Hz, 2H), 3.42 –3.40(m,4H),3.32–3.28(m,8H),2.45(s,3H),2.41(s,3H),2.27(t,J=9.8Hz,2H),1.63-1.53(m,4H),1.32(m,2H).

[0417] Step 2: Preparation of Compound N-14

[0418]

[0419] Add intermediate N-13 (200 mg, 0.32 mmol), phenol (149 mg, 1.59 mmol), 33% HBr / acetic acid (5 mL) to a 25 mL reaction bottle, stir at room temperature for 20 h, monitor the disappearance of N-13 by LCMS, and the reaction is complete. Add methyl tert-butyl ether (30 mL) directly to the reaction solution, precipitate solid, filter, and obtain 140 mg brown solid, with a yield of 92%.

[0420] Step 3: Preparation of Compound N-15

[0421]

[0422] Add intermediate N-14 (140 mg, 0.29 mmol), tert-butyl bromoacetate (130 mg, 0.67 mmol), potassium carbonate (160 mg, 1.16 mmol), N,N-dimethylformamide (5 mL) to a 25 mL reaction bottle, stir at room temperature for 20 h, and monitor the disappearance of N-14 by LCMS, indicating the completion of the reaction. Filter the reaction solution, concentrate, and perform reverse phase preparation to obtain 120 mg of an oily liquid with a yield of 75%.

[0423] 1 H NMR (400MHz, CDCl3) δ7.84 (s, 1H), 7.73 (s, 1H), 4.05 (t, J = 10.2Hz, 2H), 3.32–3.30 (m, 8H), 2. 62(m,4H),2.46(m,4H),2.29(t,J=10.1Hz,2H),1.64-1.52(m,4H),1.42(s,18H),1.31(m,2H).

[0424] Step 4: Preparation of Compound N-16

[0425]

[0426] N-15 (120 mg, 0.22 mmol) was added to a 10 mL reaction bottle. Trifluoroacetic acid (3 mL) and dichloromethane (3 mL) were added. The reaction was stirred at room temperature for 4 h. LCMS monitored the complete reaction of the raw material. The excess solvent was removed by rotary evaporation under reduced pressure. A small amount of methanol was added and 60 mg of oily liquid was obtained by reverse phase preparation. The yield was 63%.

[0427] Step 5: Preparation of Compound XIy

[0428]

[0429] Add intermediate N-16 (4 mg, 0.008 mmol), B (15 mg, 0.018 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (3 mg, 0.024 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (7 mg, 0.018 mmol) to a 25 mL reaction bottle, stir at room temperature for 20 min, monitor N-16 disappearance, reaction complete. Direct reverse phase preparation to obtain 10 mg of yellow solid, yield 58%.

[0430] Example 27. Preparation of Antibody-Drug Conjugate (ADC)

[0431] 1. Preparation

[0432] Step (1) Antibody reduction: Replace the antibody medium with pH 7.2 PBS / EDTA to prepare an antibody concentration of 4 mg / ml. Place this solution (0.5 mL) in a 1.5 mL polypropylene test tube, add TCEPPBS solution (8 ul: 3 equivalents per antibody molecule) to the solution, and incubate at 25°C for 1 hour to partially reduce the disulfide bonds in the hinge of the antibody.

[0433] Step (2) Coupling of antibody and drug linker: At 25°C, add DMSO (43uL) and DMSO solution containing the dual-drug linker assembly unit obtained in Examples 1 to 26 (6.7uL: 5 equivalents relative to one molecule of antibody) to the above solution, mix using a test tube mixer, stir at room temperature for 120 minutes to connect the drug linker to the antibody. Next, add 100mM NAC aqueous solution (2uL: 18.4 equivalents relative to one molecule of antibody), and stir at room temperature for 20 minutes to terminate the reaction of the drug linker to obtain each ADC: XIa to XIy.

[0434] 2. Characterization

[0435] The aggregation degree of ADC was detected by size exclusion chromatography, the DAR value of ADC was detected by LCMS, and the content of naked antibody in ADC was detected by HIC method.

[0436] Table 1. Characterization results of antibody-drug conjugates XIa to XIy

[0437]

[0438]

[0439] The beneficial effects of the present invention are demonstrated by test examples below.

[0440] Test Example 1: Cell Viability Test

[0441] 1. Experimental Methods

[0442] Experimental cells: N87 cells, SK-BR-3 cells, MDA-MB-468 cells.

[0443] Experimental steps: Collect cells in the logarithmic growth phase, count them using a cell counter, resuspend the cells with complete culture medium, calculate the cell concentration to the appropriate concentration and inoculate the cell suspension in a 96-well plate at 100 μl / well, and incubate in a 37°C, 100% relative humidity, 5% CO2 incubator for 24 hours. Before adding the drug, dilute the ADC to be tested with culture medium to the corresponding action concentration set, and add it to the 96-well plate at 25 μl / well. Incubate the cells in a 37°C, 100% relative humidity, 5% CO2 incubator for 144 hours. Add 10 μL / well CCK-8 and incubate in a 37°C incubator for 2-4 hours. After gently shaking, measure the absorbance at a wavelength of 450 nm on the SpectraMax i3X Reader, use the absorbance at 650 nm as a reference (i.e., absorbance at 450 nm - absorbance at 650 nm), calculate the inhibition rate, and further calculate the IC 50 value.

[0444] The known ADC on the market: Ds-8201 was used as a positive control.

[0445] 2. Experimental results

[0446] Table 2. Cytotoxicity test results

[0447]

[0448]

[0449] The experimental results show that the ADC of the present invention has good inhibitory activity against HER2-positive cell lines N87 and SK-BR-3, but has no inhibitory activity against the HER2-negative cell line MDA-MB-468, indicating that the ADC of the present invention has good anti-tumor effect and antigen specificity.

[0450] In addition, compared with the ADC Ds-8201 that has been on the market, the ADC provided by the present invention also has significantly improved inhibitory effects on HER2-positive cell lines N87 and SK-BR-3d.

[0451] In summary, the present invention provides a dual-drug link assembly unit, which can be linked with a targeting linker to obtain a corresponding dual-drug targeting linker-drug conjugate, and the dual-drug targeting linker-drug conjugate can target tumor cells, reduce toxic side effects on normal cells, and effectively overcome drug resistance, thereby achieving a synergistic anti-tumor effect. Compared with the ADC Ds-8201 that has been on the market, the ADC provided by the present invention has significantly improved inhibitory effects on HER2-positive cell lines N87 and SK-BR-3d. The dual-drug link assembly unit and dual-drug targeting linker-drug conjugate of the present invention have broad application prospects in the preparation of anti-tumor drugs for prevention and / or treatment.

Claims

1. A dual drug linker assembly unit, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: The structure of the dual-drug linkage assembly unit is one of the following structures: wherein n and p are independently selected from integers of 0 to 20, and n and p are not 0 at the same time; W2 and W3 are independently selected from methylene, And at least one of W2 and W3 is L b , L d Each independently selected from none or ethylene; D1 and D2 are the first drug structural unit and the second drug structural unit, respectively. The structures of D1 and D2 are the same or different.

2. The dual drug link assembly unit according to claim 1, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The D1 and D2 are independently selected from a drug unit targeting DNA or a drug unit targeting tubulin.

3. The dual drug linker assembly unit according to claim 2, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The drug unit targeting DNA is SN-38, Dxd, Dx-8951, and / or the drug unit targeting tubulin is Eribulin, MMAE, MMAF, maytansine.

4. A dual-drug linker assembly unit, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: The dual-drug linkage assembly unit is one of the following structures:

5. A dual-drug targeting linker-drug conjugate molecule, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: The dual-drug targeting linker-drug conjugate molecule is obtained by connecting a targeting linker and q dual-drug linker assembly units according to any one of claims 1 to 4; the targeting linker is a substance that can target and bind to the lesion site; the structure of the dual-drug targeting linker-drug conjugate molecule is shown in Formula I: in, The structure is a dual-drug link assembly unit as described in any one of claims 1 to 4; Ab is a targeting linker, 1≤q≤8.

6. The dual-drug targeting linker-drug conjugate molecule according to claim 5, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The targeting linker is an antibody, an antibody fragment, a protein, a polypeptide or a nucleic acid aptamer.

7. The dual-drug targeting linker-drug conjugate molecule according to claim 6, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The antibodies are antibodies against cell surface receptors and tumor-associated antigens.

8. A dual-drug targeting linker-drug conjugate molecule, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: The structure of the dual-drug targeting linker-drug conjugate molecule is selected from one of the following structures: Wherein, Ab is a targeting linker, 1≤q≤8.

9. The dual-drug targeting linker-drug conjugate molecule according to claim 8, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The targeting linker is an antibody, an antibody fragment, a protein, a polypeptide or a nucleic acid aptamer.

10. The dual-drug targeting linker-drug conjugate molecule according to claim 9, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The antibodies are antibodies against cell surface receptors and tumor-associated antigens.

11. A dual-drug targeting linker-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, or a deuterated compound thereof, characterized in that: The dual-drug targeting linker-drug conjugate is obtained by connecting a targeting linker and the dual-drug linking assembly unit according to any one of claims 1 to 4; the targeting linker is a substance that can target and bind to the lesion site.

12. The dual-drug targeting linker-drug conjugate according to claim 11, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The targeting linker is an antibody, an antibody fragment, a protein or a nucleic acid aptamer.

13. The dual-drug targeting linker-drug conjugate according to claim 12, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The antibodies are antibodies against cell surface receptors and tumor-associated antigens.

14. The dual-drug targeting linker-drug conjugate according to any one of claims 11 to 13, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules of formula IIa in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIb in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIc in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IId in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIe in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.6±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIf of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIg in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIh in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIi in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.7±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIj of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.8±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIk of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II1 of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIm of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.4±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIn of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula IIo of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIp in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIq in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.8±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIr in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIs of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIt in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 5.3±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIu in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIv of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIw of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIx in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9±1.0; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIy in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.8±1.

0.

15. The dual-drug targeting linker-drug conjugate according to claim 14, or its stereoisomer, or its optical isomer, or its deuterated compound, characterized in that: The dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules of formula IIa in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIb in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIc in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IId in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIe in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.6; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIf of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIg of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIh in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.1; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIi in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.7; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIj of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.8; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIk of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II1 of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIm of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.4; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIn of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.7; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in Formula IIo of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIp in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.5; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIq in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.8; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIr in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIs of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.2; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIt in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 5.3; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIu in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.5; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIv of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIw of claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.8; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIx in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.9; Or, the dual-drug targeting linker-drug conjugate is composed of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IIy in claim 8, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.

8.

16. A method for preparing the dual-drug targeting linker-drug conjugate molecule according to any one of claims 5 to 10, or the dual-drug targeting linker-drug conjugate according to any one of claims 11 to 15, characterized in that: The method comprises the following steps: The targeting linker is coupled to the dual-drug linker assembly unit to obtain the product.

17. A drug for preventing and / or treating tumors, characterized in that: It is a preparation made of the dual-drug targeting linker-drug conjugate molecule described in any one of claims 5 to 10 or the dual-drug targeting linker-drug conjugate described in any one of claims 11 to 15 as an active ingredient and pharmaceutically acceptable excipients.

18. Use of the dual-drug targeting linker-drug conjugate molecule according to any one of claims 5 to 10 or the dual-drug targeting linker-drug conjugate according to any one of claims 11 to 15 in the preparation of a drug for preventing and / or treating tumors.

19. The use according to claim 18, characterized in that: The tumor is a HER2 positive tumor.

20. The use according to claim 18 or 19, characterized in that: The tumor is selected from lung cancer, urethral cancer, colorectal cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumors, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma or sarcoma.

Images