WO2019149116A1

WO2019149116A1 - Method for preparing conjugate

Info

Publication number: WO2019149116A1
Application number: PCT/CN2019/072762
Authority: WO
Inventors: 宋帅; 田强; 唐祖建; 邓汉文; 汪静; 刘登念; 胡瑞斌; 肖亮; 薛彤彤; 蔡家强; 王利春; 王晶翼
Original assignee: 四川科伦博泰生物医药股份有限公司
Priority date: 2018-01-30
Filing date: 2019-01-23
Publication date: 2019-08-08
Also published as: CN111447939A; CN110090308A; CN110090308B

Abstract

A method for preparing a targeted medicament-bioactive molecule conjugate, a product prepared per the method, and uses of the product in preventing and/or treating abnormal cell activity-related neoplastic diseases (including but not limited to cancerous diseases).

Description

Method of preparing a conjugate

Field of invention

The invention belongs to the field of medical technology. In particular, the present invention relates to a method of preparing a targeted drug-biologically active molecule conjugate, a product prepared by the method, and a proliferative disease associated with the prevention and/or treatment of abnormalities in cellular activity (including but Use in not limited to cancer diseases).

Background of the invention

Chemotherapy was once the standard treatment for cancer, but high-killing bioactive molecules can kill normal cells, causing serious side effects. Targeted anti-tumor drugs have become a hot spot in the field of cancer research due to their simultaneous targeting and anti-tumor activity. Since the 20th century, breakthroughs have been made in the use of biomacromolecules (such as therapeutic antibodies or antibody fragments) and targeting small molecule ligands for the development of antitumor drugs and tumor targeted therapy. However, biomacromolecules, although highly targeted, have limited therapeutic effects on solid tumors; while bioactive molecules have high killing efficacy against cancer cells, they often lack targeting and often accidentally injure normal cells, causing serious toxic side effect.

In recent years, studies have found that therapeutic antibodies can be linked to biologically active molecules to form antibody-drug conjugates (ADCs). The ADC combines the targeting of antibodies with the high activity of biologically active molecules to become a "bio-missile". The antibody directs the ADC to bind to the target cell, which is then internalized by the cell, releasing the drug to treat the disease. Because the antibody has specificity and targeting to tumor cell related targets, its application value is not only reflected in the treatment, but also becomes an ideal carrier for drug targeted delivery, which reduces the side effects of the drug. Small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs) are designed with the same principle of chemically binding biologically active molecules and small molecule ligands that selectively bind to tumor cell surface receptors. Coupling, thereby increasing the targeting of effector molecules to tumor cells. The chemical structure of SMDC is almost identical to that of ADC, but small molecule ligands are used instead of antibodies. Currently, there is no SMDC listed.

Currently, there are four ADCs already on the market: Mylotarg (Gemtuzumab Ozogamicin, gemtuzumab oxazoic acid), Adcetris (Brentuximab Vedotin, CD30 mAb-MMAE), Kadcyla (Trastuzumab Emtansine, trastuzumab-美Dessert alkaloids and Besponsa (Inotuzumab ozogamicin, CD22 mAb-caccimycin).

Typically, ADC drugs consist of antibodies, biologically active molecules, and linkers. The biologically active molecule is covalently coupled to the antibody via a linker. Antibodies (eg, monoclonal antibodies) are capable of specifically recognizing specific targets on the surface of cancer cells, thereby directing the ADC to the surface of the cancer cells and allowing the ADC to enter the cancer cells through the endocytosis effect. Bioactive molecules can be released intracellularly, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

Lysine is the most common attachment site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond, which is currently the most commonly used coupling technique. With the deepening of research, there has been a technology that can achieve site-directed coupling, that is, the carboxyl group of the linker is activated by pentafluorophenol, and an amide bond is formed with the lysine ε-amino group in the antibody to complete the bioactive molecule and the antibody. Union. However, this coupling technique also has some disadvantages. The pentafluorophenol ester obtained by the activation of the carboxyl group has high reactivity, poor chemical stability, and cannot be stably existed even in some specific structures, resulting in the inability of the coupling reaction to proceed, thereby making the ADC couple The efficiency is extremely low and cannot even be obtained.

Summary of invention

The quaternary ammonium salt or oxynitride structure has good physicochemical properties (such as hydrophilicity), which greatly improves the pharmacokinetic properties of the ADC, and the hydrophilicity of the ADC having a quaternary ammonium salt or an oxynitride structure is improved. It is beneficial to reduce the cross-linking of the antibody part, thereby helping to reduce the toxicity of the ADC drug. In addition, in the preparation of the ADC drug, it is necessary to activate the carboxyl group at the terminal end of the linker by using an activating group. Since pentafluorophenol has a good leaving property, the carboxyl group is currently activated by pentafluorophenol. However, in the preparation of an ADC having a quaternary ammonium salt or an oxynitride structure, it was found that the pentafluorophenol ester containing a quaternary ammonium salt or an oxynitride structure is too reactive to be stably present, making the coupling reaction difficult to carry out, obtaining an ADC. The yield is extremely low. Through in-depth research, it was surprisingly discovered that a novel method for coupling a targeted drug to a biologically active molecule in the present invention, that is, the use of a novel carboxyl activation method greatly improves the stability of the activated carboxyl group in the linker, and successfully overcomes The coupling technique of using a pentafluorophenol to activate a carboxyl group cannot achieve the problem of stable pentafluorophenol ester, and at the same time, the coupling of the bioactive molecule with the targeted drug is achieved at a higher coupling ratio. This coupling method can be widely applied to the synthesis of targeted drug-bioactive molecular conjugates.

A first aspect of the invention provides a process for the preparation of a conjugate of formula (II),

The method comprises coupling a compound of formula (I) to a targeting drug comprising one or more amino groups (-NH ₂ ),

among them:

Each occurrence of T is independently a group of a biologically active molecule, preferably a group that inhibits or interferes with the function of the cell and/or a biologically active molecule that causes cell death or destruction, and more preferably an antitumor biologically active molecule. Group

L ₁ and L ₂ are each independently a divalent linking group at each occurrence;

Each occurrence of L ₃ is independently a cyclic or chain group comprising at least one quaternized nitrogen atom or a cyclic or chain group comprising at least one nitrogen atom substituted by =0;

Each occurrence of R ₁ is independently a substituted or unsubstituted C _1-6 alkylene group, a substituted or unsubstituted C _2-10 alkenylene group, a substituted or unsubstituted C _{2 . -10} alkynylene, substituted or unsubstituted C _3-10 cycloalkylene, substituted or unsubstituted C _6-10 arylene or substituted or unsubstituted 5-10 membered hetero Aryl;

Z is an oxygen atom or a sulfur atom, and is preferably an oxygen atom;

R _{2 is} selected from the group consisting of C _1-6 alkyl, C _3-10 cycloalkyl, 5-10 membered heterocyclyl, C _6-10 aryl and 5-10 substituted with one or more R _a Metaheteroaryl;

R _a is halogen (for example, fluorine, chlorine or bromine), cyano, nitro or trifluoromethyl, provided that when R _a is fluorine, R ₂ is not a perfluorophenyl group;

A is a group obtained by removing α amino groups in a targeted drug, and the targeted drug includes a macromolecule and a small molecule having a targeting effect, such as an antibody, DNA, RNA or a small molecule ligand;

m, n and r are each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 at each occurrence; preferably, m and n are each present at each occurrence Independently 0, 1, 2 or 3, and r is each independently 0, 1 or 2 at each occurrence; more preferably, m and n are 1 and r is 0;

α is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, α is 1, 2, 3 or 4; more preferably, α is 1 or 2.

A second aspect of the invention provides a conjugate of the formula (II), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, the coupling of the formula (II) Prepared by the above method,

Each of the groups is as defined above.

A third aspect of the invention provides a process for the preparation of a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, which process comprises a formula (I) -0) a compound which is reacted with R ₂ -ZH to give a compound of the formula (I),

Each of the groups is as defined above.

A fourth aspect of the invention provides a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,

Each of the groups is as defined above.

A fifth aspect of the invention provides a pharmaceutical composition comprising a conjugate of the formula (II) according to the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, and a pharmaceutically acceptable salt thereof One or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more additional anticancer agents such as chemotherapeutic agents and/or antibodies. The pharmaceutical composition is preferably a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.

A sixth aspect of the present invention provides a conjugate of the formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof for use in the preparation or prevention of cancer Use in medicines for diseases.

A seventh aspect of the invention provides a conjugate of the formula (II) or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof according to the invention for use in the prevention or treatment of cancer disease.

An eighth aspect of the invention provides a method of preventing or treating a cancer disease, which comprises administering to an individual in need thereof an effective amount of a conjugate of the formula (II) according to the invention or a pharmaceutically acceptable salt thereof , stereoisomers or metabolites or solvates thereof.

DRAWINGS

Figure 1 is a hydrophobic interaction chromatography (HIC) analysis map of naked anti-resistance.

Figure 2 is a HIC analysis map of Compound 2 coupled to an antibody.

Figure 3 is a HIC analysis map of Compound 7 coupled to an antibody.

Figure 4 is a HIC analysis map of Compound 8 coupled to an antibody.

Figure 5 is a HIC analysis map of Compound 9 coupled to an antibody.

Figure 6 is a HIC analysis map of Compound 10 coupled to an antibody.

Figure 7 is a HIC analysis map of Compound 11 coupled to an antibody.

Figure 8 is a liquid chromatography-mass spectrometry (LCMS) pattern of compound 2 coupled to an antibody.

Figure 9 is an LCMS map of Compound 7 coupled to an antibody.

Detailed description of the invention

definition

In the present invention, the terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise stated. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all routine steps widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

In the present invention, the term "conjugate" refers to a substance obtained by linking a biologically active molecule to a targeted drug. In some embodiments of the invention, the bioactive molecule is linked to the targeted drug via a linker. The linker is capable of cleavage in a particular environment (eg, an intracellular low pH environment) or a specific action (eg, the action of a lysosomal protease) to separate the biologically active molecule from the targeted drug. In some embodiments of the invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the invention, the bioactive molecule is linked directly to the targeted drug by a covalent bond that is capable of cleavage under a particular environment or action, thereby separating the biologically active molecule from the targeted drug.

In the present invention, the term "biologically active molecule" refers to a substance that inhibits or interferes with the function of a cell and/or causes cell death or destruction. In some embodiments of the present invention, the bioactive molecule in the conjugate is a molecule having antitumor biological activity, specifically, for example, a radioisotope such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , radioactive isotope of Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² or Lu; metal complexes such as metal platinum complexes (such as oxaliplatin) or metal gold complexes; glycopeptide antibiotics such as bleomycin Or pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan , belototecan or rupibine) or topoisomerase II inhibitors (such as actinomycin D, doxorubicin, doxorubicin, doxymimethine, daunorubicin, mitoxantrone, Podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, guanidine, pentastatin, fludarabine, cladribine or Nairabin et al; drugs that act on structural proteins, such as tubulin inhibitors (such as periwinkle) Alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel); tumor signaling pathway inhibitors such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate Acid kinase inhibitor or histidine kinase inhibitor; proteasome inhibitor; histone deacetylase inhibitor; tumor angiogenesis inhibitor; cyclin inhibitor; maytansine derivative; Derivatives; auristatin derivatives; pyrrolobenzodiazepines

Dimer (PBD) derivatives; melphalan; mitomycin C; chlorambucil or other biologically active molecules that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis; enzymes and fragments thereof, such as nucleus Lysin; antibiotic; toxin, such as a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; or growth inhibitors and the like. The term "toxin" refers to a substance that is capable of producing a detrimental effect on the growth or proliferation of cells.

A "small molecule" is defined herein as a biologically active small molecule drug. In certain embodiments, the small molecule has a molecular weight of no greater than 2000 Da, such as no greater than 1500 Da, 1000 Da, or 500 Da.

In the present invention, the term "linker" refers to a structural fragment that links a biologically active molecule to a targeted drug.

In the present invention, the term "targeting drug" refers to a drug capable of specifically binding to a target (or a portion of a target) on a cell surface. By targeting the interaction of the drug moiety with the target, the conjugate can be delivered to a particular cell population.

In the present invention, when the targeting drug is an antibody, the conjugate may be referred to as an "antibody-drug conjugate." In the present invention, "antibody-drug conjugate" and "immunoconjugate" are used interchangeably.

In the present invention, the term "antibody" is used in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, as long as they have The required biological activity. As used herein, "antibody" and "immunoglobulin" are used interchangeably.

In the present invention, the term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the cluster are identical except for a small number of natural mutations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of the antigen, while polyclonal antibodies relative thereto contain different antibodies for different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being free from contamination by other antibodies during synthesis. The modifier "monoclonal" as used herein means that the antibody is characterized by a substantially homogeneous population of antibodies and is not to be construed as being prepared by a particular method.

In some embodiments of the invention, the monoclonal antibody also specifically includes a chimeric antibody, ie, a portion of the heavy and/or light chain is identical or homologous to a certain, certain or a subset of antibodies, and the remainder is One, another or another subclass of antibodies are identical or homologous as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies useful in the present invention include primatized antibodies comprising variable region antigen binding sequences from human non-human primates (e.g., ancient monkeys or orangutans, etc.) and human constant region sequences.

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

The term "bispecific antibody" is used interchangeably with "bifunctional antibody conjugate" and refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) by a coupling arm. The conjugate retains the activity of the respective antibody and is therefore bifunctional and bispecific.

The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies having three different antigen binding specificities and the latter being antibodies having four different antigen binding specificities.

The term "intact antibody" refers to an antibody comprising an antigen binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region can be a native sequence (eg, a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

The term "Probody" is a modified antibody comprising an antibody or an antibody fragment that specifically binds to its target and is capable of coupling to a masking group, wherein the masking group refers to an antibody or antibody fragment thereof The cleavage constant of the binding ability of the target is at least 100-fold, 1000-fold or 10,000-fold greater than the cleavage constant of the binding ability of the antibody or antibody fragment without the coupling masking group to its target.

In the present invention, a "humanized" form of a non-human (eg, murine) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequences. Most humanized antibodies are human receptors. The hypervariable region residues of immunoglobulins are replaced by non-humans (eg, mice, rats, rabbits, or non-human primates) with the desired specificity, affinity, and function. Hypervariable region residues (donor antibodies). In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally comprise at least one, usually two variable regions, wherein all or nearly all of the hypervanable loops correspond to non-human immunoglobulins, while FR is completely or almost entirely human immunoglobulin. The sequence of the protein. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details, see, for example, Jones et al, 1986, Nature, 321 :522-525; Riechmann et al, 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

Intact antibodies can be divided into different "classes" based on the amino acid sequence of the heavy chain constant region. The main five classes are IgA, IgD, IgE, IgG and IgM, several of which can also be divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions of different classes of antibodies are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

In the present invention, although the amino acid substitution in the antibody is substituted by the L-amino acid in most cases, it is not limited thereto. In some embodiments, one or more D-amino acids can be included in the antibody peptide chain. Peptides comprising D-amino acids are more stable and less susceptible to degradation in the oral, intestinal or plasma than peptides comprising only L-amino acids.

The monoclonal antibodies used in the present invention can be produced by a number of methods. For example, monoclonal antibodies for use in the present invention can be obtained by hybridoma methods using a variety of species including cells of mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495). Alternatively by recombinant DNA techniques (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al, 1991, Nature, 352: 624-628; and Marks et al, 1991, Journal of Molecular Biology , 222: 581-597). Monoclonal antibodies useful in the present invention include, but are not limited to, monoclonal antibodies against Her2, such as trastuzumab or pertuzumab, or monoclonal antibodies against Trop-2, such as Sacituzumab (ie Isactuzumab or hRS7 antibody).

In some embodiments of the invention, the targeted drug is a targeted drug that targets the following targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEACAM5 , mucin (such as mucin 1 and mucin 16), CD138, CD20, CD19, CD30, SLTRK6, connexin 4, tissue factor, endothelin receptor, STEAP1, SLC39A6, guanylate cyclase C, PSMA, CCD79b, CD22, sodium phosphate cotransporter 2B, GPNMB, trophoblastic glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, short proteoglycan, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, sealing protein 18.2, BMPR1B, Tyro7, c-Met , ApoE, CD1lc, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15 , MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3 , PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43 and NaPi2b.

In some embodiments of the invention, the targeted drug is selected from the group consisting of: an RGD peptide that recognizes a cell surface integrin receptor; a growth factor that recognizes a cell surface growth factor receptor such as EGF, PDGF, or VEGF; A peptide on the cell surface plasminogen activator, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptor.

In some embodiments of the invention, the targeted drug is selected from the group consisting of: CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, DR5 ligand And connecting two aliphatic anthraquinone polyenes, cyanine dyes, IR-783 or derivatives thereof.

In some embodiments of the invention, the targeted drug is selected from the group consisting of: an anti-CEACAM5 antibody, an anti-CD22 antibody, an anti-CD20 antibody, and an anti-mucin antibody.

In some embodiments of the invention, the targeted drug is a monoclonal antibody against Her2, such as trastuzumab, pertuzumab; or the targeted drug is a monoclonal antibody against Trop-2 , for example, Sacituzumab.

In some embodiments of the invention, the targeted drug is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her2 whose amino acid sequence is known to those skilled in the art, the schematic sequence of which can be found, for example, in CN103319599. Lys at the end of the heavy chain of trastuzumab is readily deleted, but this deletion does not affect biological activity, see Dick, L. W. et al, Biotechnol. Bioeng., 100: 1132 - 1143. Exemplary heavy chain and light chain sequences of pertuzumab can be found in SEQ ID No. 16 and SEQ ID No. 15 of US7560111.

In some embodiments of the invention, the targeted drug anti-Trop-2 antibody is RS7 as described in U.S. Patent No. 7,517,964; and hRS7 (i.e., Sacituzumab of the present invention) as described in US 2012/0237518. Anti-Trop-2 antibodies useful in the present invention can also be obtained by screening methods for designing, constructing, and constructing antibody libraries displaying antibodies, as disclosed in CN103476941A.

In the present invention, ErbB2 and Her2 are used interchangeably, both of which represent the human HER2 protein of the native sequence (Genebank accession number: X03363, see, for example, Semba et al., 1985, PNAS, 82: 6497-6501; and Yamamoto et al. , 1986, Nature, 319: 230-234) and functional derivatives thereof, such as amino acid sequence variants. ErbB2 represents the gene encoding human Her2, and neu represents the gene encoding rat pl85neu. In some embodiments, the compounds or conjugates of the invention are capable of inhibiting or killing cells expressing the ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, Renal cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells or liver cancer cells.

In the present invention, Trop-2 or TROP2 refers to human trophoblast cell surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is composed of many humans. A cell surface receptor expressed by a tumor (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, or cervical cancer). In some embodiments, a compound or conjugate of the invention is capable of inhibiting or killing a cell that expresses a TROP2 receptor, such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, or cervical cancer.

In the present invention, the term "C _1-6 alkyl" means a straight or branched alkyl group having 1 to 6 carbon atoms, and includes, for example, "C _1-4 alkyl" or "C _1-3 alkyl". Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2- Methyl butyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethyl Butyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethyl Butyl, 2-ethylbutyl and 1,2-dimethylpropyl, and the like. The term "C _1-6 alkylene" is a corresponding divalent group, and includes, for example, "C _1-4 alkylene", "C _1-3 alkylene", and the like, and specific examples include, but are not limited to, Base, ethylene, propylene, butylene, pentylene and hexylene.

In the present invention, the term "C _2-10 alkenyl" means a straight-chain, branched or cyclic alkenyl group having at least one double bond and having 2 to 10 carbon atoms, and includes, for example, "C _2-6 alkene". ",""C _2-4 alkenyl" and the like. Examples thereof include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexyl Dienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, and 1,4-cyclohexadienyl, and the like. The term "C _2-10 alkenylene" is a corresponding divalent group including, for example, "C _2-6 alkenylene", "C _2-4 alkenylene" and the like. Examples thereof include, but are not limited to, a vinylidene group, a propylene group, a butenylene group, a pentenylene group, a hexylene group, a cyclopentylene group, a cyclohexylene group, and the like.

In the present invention, the term "C _2-10 alkynyl" means a straight or branched alkynyl group having at least one triple bond and having 2 to 10 carbon atoms, and includes, for example, "C _2-6 alkynyl group", "C _2-4 alkynyl" and the like. Examples thereof include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 - hexynyl and 5-methyl-2-hexynyl and the like. The term "C _2-10 alkynylene" is a corresponding divalent group including, for example, "C _2-6 alkynylene", "C _2-4 alkynylene" and the like. Examples thereof include, but are not limited to, ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

In the present invention, the term "halogen" includes fluorine, chlorine, bromine and iodine.

In the present invention, the term "3-10 membered cycloalkyl" or "C _3-10 cycloalkyl" means a saturated cyclic alkyl group having 3 to 10 carbon atoms, and includes, for example, "C _3-8 cycloalkane". ",""C _3-6 cycloalkyl", "C _4-6 cycloalkyl", "C _5-7 cycloalkyl" or "C _5-6 cycloalkyl" and the like. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The term "3-10 membered cycloalkylene" or "C _3-10 cycloalkylene" is a corresponding divalent group including, for example, "C _3-8 cycloalkylene", "C _3-6 cyclo ring"Alkyl","C _4-6 cycloalkylene", "C _5-7 cycloalkylene" or "C _5-6 cycloalkylene" and the like. Specific examples include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.

In the present invention, the term "3-8 membered heterocyclic group" means a cyclic group having 3 to 8 ring atoms in which at least one ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "3-8 membered heterocyclic group" includes, for example, "3-8 membered nitrogen-containing heterocyclic group", "3-8 membered oxygen-containing heterocyclic group", "3-6 membered heterocyclic group", and "3-6 member". "Oxygen-containing heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered heterocyclic group", "5- 6-membered nitrogen-containing heterocyclic group", preferably including but not limited to oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and gaoprene Azinyl and the like. The term "3-8 membered heterocyclylene" is a corresponding divalent group and includes, for example, "3-8 membered nitrogen-containing heterocyclylene", "3-8 membered oxygen-containing heterocyclylene", "3- 6-membered heterocyclylene", "3-6-membered oxygen-containing heterocyclylene", "4-7-membered heterocyclylene", "4-6-membered heterocyclylene", "5-7-membered heteropoly" "Cyclo", "5-6 membered heterocyclylene", "5-6 membered nitrogen-containing heterocyclylene", preferably including but not limited to ethylene oxide, oxycyclobutane, pyrrole Alkyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and xagopiperazinyl, and the like.

In the present invention, the term "6-12 membered spiroheterocyclyl" means a group of 6 to 12 ring atoms (having at least one ring atom) formed by two or more ring structures sharing one ring atom with each other. It is a cyclic structure of a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered spiroheterocyclyl" includes, for example, "6-12 membered nitrogen-containing spiroheterocyclyl", "6-11 membered spiroheterocyclyl", "6-10 membered spiroheterocyclyl", and "7- 11-membered spiroheterocyclyl", "7-10-membered spiroheterocyclyl", "7-9-membered spiroheterocyclyl", "7-8-membered spiroheterocyclyl", and "9-10-membered spiroheterocyclyl" And "8-10 yuan spiroheterocyclyl" and the like. Specific examples include, but are not limited to:

Wait. The term "6-12 membered spiroheterocyclyl" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing spiroheterocyclyl", "8-11 membered nitrogen-containing spiroheterocyclyl", "11-membered nitrogen-containing snail heterocyclic group", "6-11 membered snail heterocyclic group", "6-10 membered snail heterocyclic group", "7-11 membered snail heterocyclic group", "7 -10 membered snail heterocyclic group, "7-9 membered snail heterocyclic group", "7-8 membered snail heterocyclic group", "9-10 membered snail heterocyclic group" and "8-10" Yuan Yaspiroheterocyclic group" and the like.

In the present invention, the term "6-12 membered bridged heterocyclic group" means a group of 6 to 12 ring atoms formed by two or more ring structures sharing two non-adjacent ring atoms with each other ( A cyclic structure in which at least one of the ring atoms is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered bridged heterocyclic group" includes, for example, "6-12 membered nitrogen-containing bridged heterocyclic group", "6-11 membered bridged heterocyclic group", "6-9 membered bridged heterocyclic group", and "6- 10-membered bridged heterocyclic group, "7-10 membered bridged heterocyclic group", "7-9 membered bridged heterocyclic group", "7-8 membered bridged heterocyclic group", "8-membered bridged heterocyclic group", "9-10 membered bridged heterocyclic group" and "8-10 membered bridged heterocyclic group" and the like. Specific examples include, but are not limited to:

Wait. The term "6-12 membered subbridged heterocyclic group" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing ylidene heterocyclic group", "8-11 membered nitrogen-containing ylidene heterocyclic group", "8-membered nitrogen-containing subbridge heterocyclic group", "6-11 membered subbridge heterocyclic group", "6-9 membered subbridge heterocyclic group", "6-10 membered subbridged heterocyclic group", "7 -10 membered subbridge heterocyclic group", "7-9 membered subbridged heterocyclic group", "7-8 membered subbridged heterocyclic group", "8 membered subbridged heterocyclic group", "9-10 member Bridged heterocyclic group" and "8-10 membered subbridge heterocyclic group" and the like. Specific examples include but are not limited to

In the present invention, the term "6-12 membered fused heterocyclic group" means a group of 6 to 12 ring atoms formed by two or more ring structures sharing two adjacent atoms with each other (at least A ring structure in which a ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered fused heterocyclic group" includes, for example, "6-12 membered nitrogen-containing fused heterocyclic group", "6-11 membered fused heterocyclic group", "6-10 membered fused heterocyclic group", and "7- 10 membered fused heterocyclic group", "8-10 membered fused heterocyclic group", "8-10 membered nitrogen-containing fused heterocyclic group", "9-10 membered fused heterocyclic group", "9-10 membered nitrogen-containing group" A fused heterocyclic group" and a "6-12 membered oxygen-containing fused heterocyclic group" and the like. Specific examples include, but are not limited to, tetrahydroimidazo[4,5-c]pyridyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo[d][1 , 3]dioxolyl, 1,3-dihydroisobenzofuranyl, 4H-1,3-benzoxazinyl, 4,6-dihydro-1H-furo[3,4 -d]imidazolyl, 3a,4,6,6a-tetrahydro-1H-furo[3,4-d]imidazolyl, 4,6-dihydro-1H-thieno[3,4-d]imidazole , 4,6-dihydro-1H-pyrrolo[3,4-d]imidazolyl, benzimidazolylalkyl, octahydro-benzo[d]imidazolyl, decahydroquinolyl, hexahydrothieno Imidazolyl, hexahydrofurfurimidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahydrocyclopenta[c]pyrrolyl, indanyl, di Hydroisoisoindolyl, benzoxazole alkyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl and 4H- 1,3-benzoxazinyl and the like. The term "6-12 membered heterofused heterocyclic group" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing sulfene heterocyclic group", "6-11 membered heterofused heterocyclic group", "6" -10 membered fused heterocyclic group", "7-10 membered fused heterocyclic group", "8-10 membered fused heterocyclic group", "8-11 membered nitrogen-containing fused heterocyclic group", "8" a -10-membered nitrogen-containing fused heterocyclic group", an "8-membered nitrogen-containing fused heterocyclic group", a "9-10 membered fused heterocyclic group", a "9-10 membered nitrogen-containing fused heterocyclic group", and "6-12 yuan oxygen-containing fused heterocyclic group" and the like.

In the present invention, the term "aryl" means a monocyclic or polycyclic hydrocarbon group having an aromatic character such as a C _6-10 aryl group, a C _5-8 aryl group or the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl and the like. The term "arylene" is the corresponding divalent group, for example C _6-10 arylene, C _5-8 arylene, and the like. Specific examples include, but are not limited to, phenylene, naphthylene, anthracenylene, phenanthrylene, and the like.

In the present invention, the term "heteroaryl" refers to a cyclic group having an aromatic character in which at least one ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. Specific examples include, but are not limited to, a 5-10 membered heteroaryl group, a 5-10 membered nitrogen-containing heteroaryl group, a 6-10 membered oxygen-containing heteroaryl group, a 6-8 membered nitrogen-containing heteroaryl group, and a 5-8 membered oxygen group. a heteroaryl group or the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1, 2,3-Triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl 1,3,4-oxadiazolyl, pyridyl, 2-pyridinone, 4-pyridinone, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxa Azinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4- Azinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, azepanyl And 1,3-diazacycloheptatrienyl and azacyclotetradecenyl and the like. The term "heteroarylene" is a corresponding divalent group, and specific examples include, but are not limited to, nitrogen-containing heteroarylene, 5-10 membered heteroarylene, 5-10 membered nitrogen-containing heteroarylene, 5- 6-membered nitrogen-containing heteroarylene, 6-membered nitrogen-containing heteroarylene, 6-10-membered oxygen-containing heteroarylene, 6-8-membered nitrogen-containing heteroarylene, and 5-8-membered oxygen-containing heteroarylene Et., for example, a furanyl group, a thienylene group, a pyridylene group, a thiazolyl group, a oxazolyl group, an imidazolyl group, a pyrazolyl group, a striazolyl group, a pyridylene group, a pyrimidinyl group, a oxazinyl group. , pyridazinyl, pyrazine, tritriazinyl and tetrazinylene.

In the present invention, the term "counter ion" means an ion that is accompanied by an ionic species to maintain electrical neutrality, for example, in sodium chloride, a sodium cation is a counter ion of a chloride anion, and vice versa.

Optionally, the hydrogen in the groups involved in the present invention may be substituted by deuterium.

The group referred to in the present invention is obtained by substituting 1, 2 or 3 hydrogen atoms of the compound/conjugate in the group with other atoms, and the number of the substituted hydrogen atoms It can be determined based on the number of valence bonds formed by the group in the compound or conjugate of the invention. For example, an alkyl group is a group obtained by substituting one hydrogen atom in an alkane, and an alkylene group is a group obtained by substituting two hydrogen atoms in an alkane, and the methyl group and the ethyl group are respectively one of methane and ethane. The group obtained by substituting a hydrogen atom, a methylene group and an ethylene group are respectively a group obtained by substituting two hydrogen atoms in methane and ethane.

As used herein, the term "substituted" means that one or more (eg, one, two, three, or four) hydrogens on the designated atom are replaced by the selection of the indicated group, provided that the The normal valence of the specified atom in the present case and the substitution form a stable compound/conjugate. Combinations of substituents and/or variables are permissible only if such combinations form stable compounds/conjugates.

If a substituent is described as "optionally substituted," the substituent may be unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the carbon (to the extent of any hydrogen present) may be independently and/or together independently The optional substituents selected are substituted. If the nitrogen of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected. Substitute substitution.

If a substituent is described as being "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.

The substituents of the substituents, as used herein, may be derived from any suitable position of the substituent, unless otherwise indicated.

When a bond of a substituent is shown to pass through a bond connecting two atoms in the ring, such a substituent may be bonded to any of the ring-forming atoms in the substitutable ring.

As used herein, the term "solvate" refers to a substance formed by association of a compound/conjugate with a solvent molecule. The solvent may be an organic solvent (for example, methanol, ethanol, propanol, acetonitrile, etc.) or the like. For example, a compound/conjugate of the invention can form an ethanolate with ethanol.

In the present invention, the term "hydrate" means a substance formed by association of a compound/conjugate with a water molecule.

In an embodiment of the invention, if a chiral carbon is present in the compound/conjugate, the invention includes isomers formed based on any stereo configuration of the chiral carbon, for example including racemates or any Mirror isomer. Moreover, the invention includes all other stereoisomers that may be present. That is, the compounds/conjugates of the present invention include all enantiomers, diastereomers, cis and trans isomers, racemates and the like.

Solid lines can be used in this article

Solid wedge

Virtual wedge

The chemical linkages of the compounds/conjugates of the invention are depicted. The use of solid lines to delineate linkages bonded to an asymmetric carbon atom is intended to include all possible stereoisomers at the carbon atom (eg, specific enantiomers, racemic mixtures, etc.). The use of a solid or virtual wedge to characterize a bond to an asymmetric carbon atom is intended to indicate the presence of the stereoisomers shown. When present in a racemic mixture, solid and virtual wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise indicated, the compounds/conjugates of the invention are intended to be stereoisomers (including cis and trans isomers, optical isomers (eg, R and S enantiomers), diastereomeric Forms of isomers, geometric isomers, rotamers, conformers, atropisomers, and mixtures thereof exist. The compounds/conjugates of the invention may exhibit more than one type of isomerism and consist of a mixture thereof (e.g., a racemic mixture and a diastereomeric pair).

The invention encompasses all possible crystalline forms or polymorphs of the compounds/conjugates of the invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

Pharmaceutically acceptable salts of the compounds/conjugates of the invention include the acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, glucoheptonate, gluconate, orotate, palmitate, and other similar salts.

Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts, and other similar salts.

For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds/conjugates of the invention are known to those skilled in the art.

Also included within the scope of the invention are metabolites of the compounds/conjugates of the invention, ie, substances formed in vivo upon administration of the compounds/conjugates of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, and the like of the administered compound/conjugate. Accordingly, the invention includes metabolites of the compounds/conjugates of the invention, including compounds/conjugates prepared by contacting a compound/conjugate of the invention with a mammal for a time sufficient to produce a metabolic product thereof.

Preparation

In some embodiments, the invention provides a method of preparing a conjugate of formula (II),

among them:

Z is an oxygen atom or a sulfur atom, and is preferably an oxygen atom;

In a preferred embodiment, T is a group selected from the group consisting of a metal complex such as a metal platinum complex (such as oxaliplatin) or a metal gold complex; a glycopeptide antibiotic such as Boramycin Or pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topological Kang, belototecan or rupibine) or topoisomerase II inhibitors (such as actinomycin D, doxorubicin, doxorubicin, doxymimethine, daunorubicin, mitoxantrone , podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, guanidine, pentastatin, fludarabine, cladribine Or nairabin, etc.; drugs acting on structural proteins, such as tubulin inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel); tumor signaling pathways Inhibitors such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate Kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; maytansin derivatives; calicheamicin Derivative; auristatin derivative; pyrrolobenzodiazepine

Dimer (PBD) derivatives; melphalan; mitomycin C; and chlorambucil or other biologically active molecules that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis.

Preferably, T is selected from the following structures:

More preferably, T is selected from the following structures:

In a preferred embodiment, L _{1 is} selected from the group consisting of an amino acid group, a peptide group, an oligosaccharide group, -(CH ₂ ) _t -, -(CH ₂ CH ₂ O) _t -(CH ₂ ) _t -, -(CH ₂ ) _t -(CH ₂ CH ₂ O) _t -,

The group is linked to T by one of the two positions labeled 1 or 2 and to L ₂ by another position; preferably, the group is linked to T by a position of 1 mark and is marked by 2 Position connected to L ₂ ;

among them:

R _j and R _k are each independently selected from H (hydrogen), D (oxime), halogen, -COOH, -SO ₃ H, CF ₃ , CN, CH ₂ CN, C _1-6 alkyl, C _1-6 Alkoxy, C _2-10 alkenyl, C _2-10 alkynyl and C _3-6 cycloalkyl;

t each independently is an integer of 1, 2, 3, 4, 5 or 6 at each occurrence;

x is an integer of 0, 1, 2, 3, 4, 5 or 6;

z is an integer of 0, 1, 2 or 3.

Preferably, L _{1 is} selected from the group consisting of a peptide group consisting of 2-5 amino acids, an oligosaccharide group,

The group is linked to T by one of the two positions labeled 1 or 2 and to L ₂ by another position; preferably, the group is linked to T by a position of 1 mark and is marked by 2 The location is connected to L ₂ .

More preferably, L ₁ is

In a preferred embodiment, L ₂ is a group selected from the group consisting of an amino acid group, a peptide group of 2-10 amino acids, an oligosaccharide group, -(CH ₂ ) _y -, -(CH ₂ CH ₂ O) _y -(CH ₂ ) _y -, -(CH ₂ ) _y -(CH ₂ CH ₂ O) _y -,

The group is linked to L ₁ by one of the two positions labeled 1 or 2 and to L ₃ by another position; preferably, the group is linked to L ₁ by the position of 1 mark, and passes through 2 The position of the mark is connected to L ₃ ;

among them:

R _j , R _k , R _m and R _n are each independently selected from H (hydrogen), D (oxime), halogen, -COOH, -SO ₃ H, carboxylic acid, sulfonic acid, CF ₃ , CN, CH ₂ CN a C _1-6 alkyl group, a C _1-6 alkoxy group, a C _2-10 alkenyl group, a C _2-10 alkynyl group, and a C _3-6 cycloalkyl group;

y is each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 at each occurrence.

Preferably, L _{2 is} selected from the group consisting of an amino acid group, a peptide group consisting of 2-5 amino acids,

The group is linked to L ₁ by one of the two positions labeled 1 or 2 and to L ₃ by another position; preferably, the group is linked to L ₁ by the position of 1 mark, and passes through 2 marked locations connected to L _3.

More preferably, L ₂ is a group selected from the group consisting of:

The group is linked to L ₁ by one of the two positions labeled 1 or 2 and to L ₃ by another position; preferably, the group is linked to L ₁ by the position of 1 mark, and passes through 2 The position of the mark is connected to L ₃ ; further preferably, L ₂ is

The group is attached to L ₁ through the position of the ₁ mark and to the L ₃ through the position of the 2 mark.

In a preferred embodiment, L _{3 is} selected from the group consisting of the following: optionally substituted by one or more R _i :

a 3-8 membered nitrogen-containing heterocyclylene group containing at least one quaternized nitrogen atom, a 6-12 membered nitrogen-containing ylidene heterocyclic group, a 6-12 membered nitrogen-containing siroheterocyclyl group, and a 6-12 member a nitrogen-containing heterocyclic heterocyclic group, a nitrogen-containing heteroarylene group, a C _3-8 cycloalkylene group-W-, and a 3-8 membered nitrogen-containing heterocyclylene group containing at least one nitrogen atom substituted by =0, wherein W is

E is a counter ion, and each of R _p is independently selected from C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 alkoxy, and -C _1-2 alkyl-cyano at each occurrence. And R _q is each independently selected from C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl and C _3-8 cycloalkyl at each occurrence.

Preferably, L _{3 is} selected from the group consisting of the following: optionally substituted by one or more R _i :

a 5-6 membered nitrogen-containing heterocyclic heterocyclic group containing at least one quaternized nitrogen atom, a 8-11 membered nitrogen-containing ylidene heterocyclic group, a 8-11 membered nitrogen-containing snail heterocyclic group, and 8-11 members a nitrogen-containing heterocyclic heterocyclic group, a 5-6 membered nitrogen-containing heteroarylene group, a C _5-6 cycloalkylene group-W-, and a 5-6 membered nitrogen-containing heteropoly group containing at least one nitrogen atom substituted by =O Ring base, where W is

Preferably, L ₃ is selected from optionally substituted with one or more substituents R _i of the following groups:

a 5-6 membered nitrogen-containing heterocyclylene group containing at least one quaternized nitrogen atom, an 8 membered nitrogen-containing ylidene heterocyclic group, a 11-membered nitrogen-containing snail heterocyclic group, and a 8-11 membered nitrogen-containing snail a cyclic group, a 5-6 membered nitrogen-containing heteroarylene (eg, pyridylene), a C _5-6 cycloalkylene-W-, and a 5-6 membered nitrogen containing at least one nitrogen atom substituted with =O Heterocyclylene, wherein W is

a 5-6 membered nitrogen-containing heterocyclic heterocyclic group containing at least one quaternized nitrogen atom, an 8 membered nitrogen-containing ylidene heterocyclic group, an 11-membered nitrogen-containing snail heterocyclic group, and an 8-membered nitrogen-containing fused heterocyclic group a 6-membered nitrogen-containing heteroarylene group, a cyclohexylene group-W-, and a 5-6 membered nitrogen-containing heterocyclylene group containing at least one nitrogen atom substituted with =O, wherein W is

E is a counterion, each of R _p is independently selected from -CH ₂ CN and -CH ₂ CH ₂ CN, and R _q is each independently selected from C _1-6 alkyl, C at each occurrence. _2-6 alkenyl, C _2-6 alkynyl and C _3-8 cycloalkyl.

More preferably, L _{3 is} selected from the group consisting of the following: optionally substituted by one or more R _i :

The group is attached to R ₁ by one of two positions labeled 1 or 2 and to L ₂ via another position; preferably, the group is linked to R ₁ by the position of 1 mark, and passes through 2 The position of the mark is connected to L ₂ ;

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion;

R _q is each independently selected from C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl and C _3-8 cycloalkyl at each occurrence;

R _p is each independently selected from C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 alkoxy and -C _1-2 alkyl-cyano, preferably -CH, in each occurrence. ₂ CN and -CH ₂ CH ₂ CN;

β is an integer of 0, 1, or 2;

γ is each independently an integer of 1, 2 or 3 at each occurrence;

R _{i is} selected from the group consisting of H (hydrogen), D (oxime), halogen, =O, CF ₃ , CN, CH ₂ CN, C _1-6 alkyl, C _1-6 alkoxy, C _2-10 alkenyl, C _3-8 cycloalkyl, C _2-10 alkynyl, COOH and SO ₃ H, preferably, R _{i is} selected from H (hydrogen), D (氘), =O, CN, CH ₂ CN, methyl and CF ₃ .

among them:

R _q is each independently selected from C _1-3 alkyl, C _2-4 alkenyl, C _2-4 alkynyl and C _3-6 cycloalkyl at each occurrence;

R _{i is} selected from the group consisting of H (hydrogen), D (oxime), CN, CH ₂ CN, methyl and CF ₃ .

More preferably, L _{3 is} selected from the group consisting of:

among them:

Further preferably, L _{3 is} selected from the group consisting of

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion.

In a preferred embodiment, R _{1 is} selected from the group consisting of C _1-6 alkylene, C _3-10 cycloalkylene, C _6-10 arylene optionally substituted by one or more R _b And a 5-10 membered heteroarylene wherein R _b is H (hydrogen), D (oxime), halogen, cyano, nitro, trifluoromethyl, COOH or SO ₃ H.

In a preferred embodiment,

From:

The group is attached to L ₁ through the position of the 1 tag and is attached to the carbonyl group via the position of the 2 tag.

Preferably,

From:

In a preferred embodiment, R _{2 is} selected from the group consisting of C _1-6 alkyl, C _3-8 cycloalkyl, 5-10 membered heteroaryl, optionally substituted by one or more R _a ,

And R _a is fluorine, chlorine, bromine, nitro or trifluoromethyl, provided that when R _a is halogen, R ₂ is not a perhalogenated C _6-10 aryl group.

Preferably, R _{2 is} selected from the group consisting of optionally substituted by one or more R _a : C _1-6 alkyl, 5-10 membered heteroaryl,

And R _a is fluorine, nitro or trifluoromethyl, provided that when R _a is fluorine, R ₂ is not a perfluoro C _6-10 aryl group.

More preferably, R _{2 is} selected from the group consisting of: C _1-6 alkyl and optionally substituted by one or more R _a

And R _a is fluorine or a nitro group, provided that when R _a is fluorine, R ₂ is not a perfluorophenyl group.

More preferably, R ₂ is a phenyl group substituted with four fluorine atoms, a phenyl group substituted with three fluorine atoms, a phenyl group substituted with two fluorine atoms, a phenyl group substituted with a fluorine atom, a phenyl group substituted with a nitro group, and a hexene group. a fluorine atom-substituted C _3-6 alkyl group, five fluorine atom-substituted C _2-6 alkyl groups, four fluorine atom-substituted C _2-6 alkyl groups or three fluorine atom-substituted C _1-6 alkyl groups .

More preferably, R _{2 is} selected from the group consisting of

Further preferably, R ₂ is 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl or 2,6-difluorophenyl.

In a preferred embodiment,

From:

Preferably,

From:

More preferably,

From:

Further preferably,

From:

In a preferred embodiment, the targeted drug is selected from the group consisting of: small molecule ligands, proteins, polypeptides, antibodies, and non-proteinaceous drugs such as sugars, RNA, or DNA.

Preferably, the targeted drug is selected from the group consisting of: a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (such as a carbonic anhydrase IX inhibitor). ; RGD peptides that recognize cell surface integrin receptors; growth factors that recognize cell surface growth factor receptors such as EGF, PDGF or VEGF; recognize functional cell surface plasminogen activator, bombesin, bradykinin , somatostatin or peptide of prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, DR5 ligand, linkage a two aliphatic steroid polyene, a cyanine dye, IR-783 or a derivative thereof; and an antibody or an active fragment or variant thereof, such as a monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or Antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, complementarity determining region fragments, single chain antibodies (eg, scFv), non-human antibodies, humanized antibodies, chimeric antibodies, all Human antibody, pro-antibody, bispecific antibody or multi-specific antibody.

Preferably, the targeted drug is a targeted drug targeting the following targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEACAM5, mucin (eg sticky Protein 1 and mucin 16), CD138, CD20, CD19, CD30, SLTRK6, connexin 4, tissue factor, endothelin receptor, STEAP1, SLC39A6, guanylate cyclase C, PSMA, CCD79b, CD22, sodium phosphate Co-transporter 2B, GPNMB, trophoblastic glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783 , STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, short proteoglycan, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, sealing protein 18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD 45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1 AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2 , IGF-1R, RNF43 and NaPi2b;

More preferably, the targeted drug is a monoclonal antibody against Her2 or an active fragment or variant thereof, or a monoclonal antibody against Trop-2 or an active fragment or variant thereof.

Further preferably, the targeted drug is trastuzumab, pertuzumab or Sacituzumab.

In a preferred embodiment, a is an integer of 1, 2, 3 or 4.

In a preferred embodiment, the method comprises mixing a targeted drug with a compound of formula (I).

Preferably, the molar ratio of the targeted drug to the compound of formula (I) is 1: (1-20).

Preferably, the method comprises mixing a solution comprising a targeted drug with a compound of formula (I).

Preferably, the process is carried out in water or an organic solvent.

Preferably, the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as Methylene chloride, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols ( Such as methanol or ethanol) and any combination thereof.

Preferably, the method further comprises purifying by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, and affinity chromatography.

In some embodiments, the invention provides a conjugate of formula (II), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,

Each of the groups is as defined above.

Preferably, the conjugate of the formula (II) is prepared by the above process.

Preferably, the conjugate of formula (II) is an antibody-drug conjugate.

Preferably, the antibody-drug conjugate is:

Wherein a is an integer of 1, 2, 3 or 4; and A is a group obtained by removing α amino groups from trastuzumab, pertuzumab or Sacituzumab.

Preferably, the antibody-drug conjugate is:

Wherein A1 is a group obtained by removing two amino groups from trastuzumab.

Preferably, the antibody-drug conjugate is:

Wherein A2 is a group obtained by removing two amino groups from pertuzumab.

Preferably, the antibody-drug conjugate is:

Wherein A3 is a group obtained by removing two amino groups in Sacituzumab.

Preferably, the antibody-drug conjugate is:

Wherein A1' is a group obtained by removing one amino group from trastuzumab.

Preferably, the antibody-drug conjugate is:

Wherein A2' is a group obtained by removing one amino group from pertuzumab.

Preferably, the antibody-drug conjugate is:

Wherein A3' is a group obtained by removing one amino group from Sacituzumab.

In some embodiments, the invention provides a method of preparing a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, the method comprising The compound of (I-0) is reacted with R ₂ -ZH to give a compound of the formula (I),

Wherein each group is as defined above;

The reaction is preferably carried out in water or an organic solvent.

Preferably, the reaction is in a base (including an organic base (such as triethylamine, DIPEA, pyridine, NMM or DMAP) or an inorganic base (such as NaH, NaOH, Na ₂ CO ₃ , NaHCO ₃ , K ₂ CO ₃ ) and It is carried out in the presence of a condensation reagent such as HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP or PyBOP.

In some embodiments, the invention provides a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,

Each of the groups is as defined above.

In a preferred embodiment, the compound of formula (I) is prepared by a process as described above.

In a preferred embodiment, the compound of formula (I) has the structure of formula (Ia),

More preferably, the compound is selected from the group consisting of

Pharmaceutical compositions and methods of treatment

In some embodiments, the present invention provides a pharmaceutical composition comprising a conjugate of Formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof And one or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more additional anticancer agents such as chemotherapeutic agents and/or antibodies. The pharmaceutical composition is preferably a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.

In some embodiments, the invention provides a pharmaceutical formulation comprising a conjugate of formula (II) according to the invention, or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, Or a pharmaceutical composition according to the invention is included.

In some embodiments, the present invention provides a conjugate of Formula (II), or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, according to the present invention, in the preparation for prophylaxis or Use in medicines for treating cancer diseases.

In some embodiments, the present invention provides a conjugate of Formula (II), or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, according to the present invention, for use in prophylaxis or Treat cancer diseases.

In some embodiments, the invention provides a method of preventing or treating a cancer disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate prepared according to the method of the invention or a pharmaceutically acceptable salt thereof , stereoisomers or metabolites or solvates thereof.

In some embodiments, the cancer disease is selected from the group consisting of esophageal cancer (eg, esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (eg, small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma , bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, salivary adenocarcinoma, colon cancer, colorectal cancer, liver cancer, kidney cancer, solid tumor, non-Hodge Gold lymphoma, central nervous system tumors (eg, glioma, glioblastoma multiforme or glioma or sarcoma), prostate cancer, and thyroid cancer.

"Pharmaceutically acceptable carrier" in the context of the present invention means a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting humans and/or within the scope of sound medical judgment. Tissues of other animals without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers for use in the pharmaceutical or pharmaceutical formulations of the invention include, but are not limited to, sterile liquids such as water and oils, including those oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, large Soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition or pharmaceutical preparation is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol and the like. The composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents as needed. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical or pharmaceutical preparation of the invention may act systemically and/or locally. For this purpose, they may be administered in a suitable route, for example by injection (for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or by oral, buccal, or oral administration. Nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

For these administration routes, the pharmaceutical composition or pharmaceutical preparation of the present invention can be administered in a suitable dosage form.

The dosage forms include, but are not limited to, tablets, capsules, troches, hard candy, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions. Injectable solutions, elixirs, syrups.

The term "effective amount" as used herein refers to the amount of a conjugate that, after administration, will alleviate one or more symptoms of the condition being treated to some extent.

The dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent need for treatment. It is noted that the dose value can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. It is to be further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or the composition of the supervised composition.

The amount of conjugate of the invention administered will depend on the severity of the individual, condition or condition being treated, the rate of administration, the handling of the conjugate, and the judgment of the prescribing physician. Generally, an effective dose will be from about 0.0001 to about 50 mg per kg body weight per day, for example from about 0.01 to about 10 mg/kg/day (single or divided doses). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, such as from about 0.7 mg/day to about 700 mg/day. In some cases, a dose level that is not higher than the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any harmful side effects, provided that the larger The dose is divided into several smaller doses to be administered throughout the day.

The conjugate of the present invention may be included in the pharmaceutical composition or pharmaceutical preparation in an amount or in an amount of from about 0.01 mg to about 1000 mg, suitably from 0.1 to 500 mg, preferably from 0.5 to 300 mg, more preferably from 1 to 150 mg, particularly preferably from 1 to 50 mg. For example, 1.5 mg, 2 mg, 4 mg, 10 mg, 25 mg, and the like.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of a condition or condition to which such a term applies or the progression of one or more symptoms of such a condition or condition, or preventing such A condition or condition or one or more symptoms of such condition or condition.

"Individual" as used herein includes human or non-human animals. Exemplary human individuals include a human individual (referred to as a patient) or a normal individual having a disease, such as the disease described herein. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (eg, birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (eg, sheep, dogs). , cats, cows, pigs, etc.).

Advantageous effects of the invention

The invention utilizes a novel carboxyl activation mode, which greatly improves the stability of the intermediate obtained by activation of the carboxyl group in the linking group, and successfully overcomes the non-pentafluorophenol ester intermediate in the presence of the quaternary ammonium salt or the oxynitride structure. Stable problems while coupling bioactive molecules to targeted drugs at a higher coupling ratio (eg, about 33%). This coupling method can be widely applied to the synthesis of targeted drug-bioactive molecular conjugates.

Example

The above content of the present invention will be further described in detail below by way of examples. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

The structure of the compound/conjugate described in the following examples was determined by nuclear magnetic resonance ( ¹ H NMR) or mass spectrometry (MS).

The nuclear magnetic resonance ( ¹ H NMR) measuring instrument uses a Bruker 400 MHz NMR spectrometer; the solvent is deuterated methanol (CD ₃ OD), deuterated chloroform (CDCl ₃ ) or hexamethyl dimethyl sulfoxide (DMSO-d). ₆ ); The internal standard substance is tetramethylsilane (TMS).

The abbreviations in the nuclear magnetic resonance (NMR) spectrum used in the examples are as follows:

s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quadruple Quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet , br: broad peak, J: coupling constant, Hz: Hertz, DMSO-d ₆ : hexamethylene dimethyl sulfoxide.

All δ values are expressed in ppm values.

The mass spectrometer (MS) assay instrument used an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Synthesis of compounds containing bioactive molecules and linkers

Example 1

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-1- Synthesis of methyl-4-((2,3,5,6-tetrafluorophenoxy)carbonyl)piperidine-1-oxime iodide (Compound 1)

step one:

Synthesis of Ethyl 1-(4-(tert-Butoxy)-4-oxobutyl)piperidine-4-carboxylate (Compound 1-2)

Compound 1-1 (1.0 g, 6.4 mmol) was dissolved in N,N-dimethylformamide (10 mL) at room temperature. To the reaction mixture was added t-butyl 4-bromobutyrate (1.7 g, 7.6 mmol) ) Potassium carbonate (1.77 g, 12.8 mmol) and potassium iodide (0.53 g, 3.2 mmol). After the addition was completed, the mixture was stirred at room temperature for 5 h, and the reaction mixture was poured into water, ethyl acetate (20 mL × 3) was evaporated, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and evaporated. Column purification (petroleum ether / ethyl acetate = 3/2) gave 1.5 g of the desired compound. ESI-MS (m/z): 300.2 [M+H] ⁺ .

Step two:

Synthesis of 1-(4-(tert-butoxy)-4-oxobutyl)-4-(ethoxycarbonyl)-1-methylpiperidine-1-oxime iodide (Compound 1-3)

Compound 1-2 (500 mg, 1.7 mmol) and iodomethane (2413 mg, 17.0 mmol) were dissolved in dichloromethane (10 mL). The solvent was evaporated under reduced pressure to give the title compound 530. ESI-MS (m/z): 314.2 [M] ⁺ .

Step three:

Synthesis of 1-(3-carboxypropyl)-4-(ethoxycarbonyl)-1-methylpiperidine-1-oxime iodide (compounds 1-4)

The compound 1-3 (530 mg, 1.7 mmol) was dissolved in dichloromethane (3 mL). The solvent was evaporated under reduced pressure to give the title compound m. ESI-MS (m/z): 258.2 [M] ⁺ .

Step four:

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of (ethoxycarbonyl)-1-methylpiperidine-1-oxime iodide (compounds 1-5)

(S)-2-((S)-2-Amino-3-methylbutyryl)-N-(4-((())((2R,3R)-3-) ((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3 -dimethylbutyrylamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropane Amido)methyl)phenyl)-5-ureidovaleramide (300 mg, 0.27 mmol) and compound 1-4 (127 mg, 0.33 mmol) dissolved in N,N-dimethylformamide (3 mL), cooled To 0 ° C, N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinium hexafluorophosphate (281 mg, 0.54 mmol) were added sequentially. After the addition, the reaction system was stirred at room temperature for 3 h. Purification by preparative liquid chromatography gave 200 mg of the title compound. ESI-MS (m/z): 1346.2 [M] ⁺ .

Step five:

4-carboxy-1-(4-(((S)-1-((())((()))) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl)-N,3-dimethyl Butyramide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido) Phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl Synthesis of 1-methylpiperidine-1-oxime iodide (compounds 1-6)

Compound 1-5 (200 mg, 0.15 mmol) was dissolved in a mixture of EtOAc (EtOAc) (EtOAc) The solution was adjusted to pH = 4 by adding a hydrochloric acid solution (0.5 mol/L), and the solvent was evaporated under reduced pressure to give purified crystals. ESI-MS (m/z): 1318.2 [M] ⁺ .

Step six:

Synthesis of Compound 1

Compound 1-6 (50 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (2 mL). Carbonyl diimide hydrochloride (11 mg, 0.06 mmol) and 4-dimethylaminopyridine (15 mg, 0.12 mmol) were cooled to 0 ° C, and 2,3,5,6-tetrafluorophenol (67 mg, 0.4 mmol), warmed to room temperature and allowed to react overnight. Purification by preparative liquid chromatography gave 5.6 mg of the title compound. ESI-MS (m/z): 1466.2 [M] ⁺ .

Example 2

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of ((2,4,6-trifluorophenoxy)carbonyl)piperidine-1-oxide (Compound 2)

step one:

Synthesis of 1-(4-(tert-butoxy)-4-oxobutyl)-4-(ethoxycarbonyl)piperidine-1-oxide (Compound 2-2)

Compound 1-2 (700 mg, 2.34 mmol) was dissolved in dichloromethane (10 mL) EtOAc. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction mixture was quenched with saturated aqueous sodium bicarbonate, extracted with dichloromethane (50 mL×3) and washed with saturated aqueous sodium hydrogen carbonate (100 mL×2). The organic phase was washed with water and dried over anhydrous sodium sulfate. ESI-MS (m/z): 316.2 [M+H] ⁺ .

Step two:

Synthesis of 1-(3-carboxypropyl)-4-(ethoxycarbonyl)piperidine-1-oxide (Compound 2-3)

The compound 2-3 was replaced by the compound 2-2 to give the title compound (500 mg). ESI-MS (m/z): 260.2 [M] ⁺ .

Step three:

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl) Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4-( Synthesis of ethoxycarbonyl)piperidine-1-oxide (compounds 2-4)

Substituting compound 2-3 for compound 1-4 using a similar procedure to that described in step 4 of Example 1 gave the title compound 15 mg. ESI-MS (m / z) : 674.5 [M / 2 + 1] +.

Step four:

4-carboxylic acid-1-(4-(((S)-1-((())(((()))) )-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl)-N,3-dimethyl Butyramido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido) Phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl Synthesis of piperidine-1-oxide (compound 2-5)

Substituting compound 2-4 for compound 1-5 to give the title compound 15 mg. ESI-MS (m/z): 660.5 [M/2+1] ⁺ .

Step five:

Synthesis of Compound 2

A similar operation as described in the first step of Example 1 was carried out, substituting compound 2-5 for compound 1-6 and 2,4,6-trifluorophenol for 2,3,5,6-tetrafluorophenol to give the title compound 2.0. Mg. ESI-MS (m/z): 725.6 [M/2+1] ⁺ .

Example 3

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-1- Synthesis of methyl-4-((2,4,6-trifluorophenoxy)carbonyl)piperidine-1-oxime iodide (compound 3)

The title compound 21 mg was obtained by substituting 2,3,5,6-tetrafluorophenol with 2,4,6-trifluorophenol. ESI-MS (m/z): 1448.2 [M] ⁺ .

Example 4

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-1- Synthesis of methyl-4-((2,2,3,3,3-pentafluoropropoxy)carbonyl)piperidine-1-oxime iodide (Compound 4)

The title compound 21 mg was obtained by substituting 2,2,3,3,3-pentafluoropropan-1-ol for 2,3,5,6-tetrafluorophenol. ESI-MS (m/z): 1450.2 [M] ⁺ .

Example 5

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of ((3-fluorophenoxy)carbonyl)-1-methylpiperidine-1-oxime iodide (Compound 5)

A similar operation as described in the first step of Example 1 was carried out, and 2-fluorophenol was used instead of 2,3,5,6-tetrafluorophenol to give the title compound 5 mg. ESI-MS (m/z): 1412.2 [M] ⁺ .

Example 6

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of ((4-fluorophenoxy)carbonyl)-1-methylpiperidine-1-oxime iodide (Compound 6)

A similar operation to that described in the first step of Example 1 was followed by substituting 4-fluorophenol for 2,3,5,6-tetrafluorophenol to give the title compound 4 mg. ESI-MS (m/z): 1412.2 [M] ⁺ .

Example 7

4-((2,3-difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidin-1-indole iodide (Compound 7)

A similar operation as described in the first step of Example 1 was carried out, and 2,3-difluorophenol was used instead of 2,3,5,6-tetrafluorophenol to give the title compound 5 mg. ESI-MS (m/z): 1430.2 [M] ⁺ .

Example 8

4-((2,5-Difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidin-1-indole iodide (Compound 8)

A similar operation as described in the first step of Example 1 was carried out, and 2,5-difluorophenol was substituted for 2,5,5,6-tetrafluorophenol to give the title compound 6 mg. ESI-MS (m/z): 1430.2 [M] ⁺ .

Example 9

4-((2,6-Difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidin-1-indole iodide (Compound 9)

A similar operation as described in the first step of Example 1 was used, and 2,6-difluorophenol was used instead of 2,3,5,6-tetrafluorophenol to give the title compound 6 mg. ESI-MS (m/z): 1430.2 [M] ⁺ .

Example 10

4-((3,4-Difluorophenoxy)carbonyl)-1-(4-(((S)-1-((S)-1-((4-((())))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidin-1-indole iodide (Compound 10)

A similar operation as described in the first step of Example 1 was carried out, and 2,4-difluorophenol was used instead of 2,3,5,6-tetrafluorophenol to give the title compound 5 mg. ESI-MS (m/z): 1430.2 [M] ⁺ .

Example 11

4-((2,3-difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)quinine-1-anthracene bromide (Compound 11)

step one:

Synthesis of 1-(4-(tert-butoxy)-4-oxobutyl)-4-(ethoxycarbonyl)quinine-1-anthracene bromide (Compound 11-2)

Compound 11-1 (500 mg, 2.7 mmol) was dissolved in dichloromethane (5 mL), and ethyl 4-bromobutyrate (602 mg, 2.7 mmol) was added to the reaction mixture. After the addition was completed, the mixture was evaporated. ESI-MS (m/z): 326.2 [M+H] ⁺ .

Step two:

Synthesis of 1-(3-carboxypropyl)-4-(ethoxycarbonyl)quinine-1-anthracene bromide (Compound 11-3)

Compound 11-2 (200 mg, 0.44 mmol) was dissolved in EtOAc (EtOAc m. The solvent was evaporated to dryness crystals crystals crystall ESI-MS (m/z): 270.2 [M] ⁺ .

Step three:

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of (ethoxycarbonyl)quinine-1-indole bromide (Compound 11-4)

(S)-2-((S)-2-Amino-3-methylbutyryl)-N-(4-((())((2R,3R)-3-) ((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3 -dimethylbutyrylamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropane Amido)methyl)phenyl)-5-ureidovaleramide (300 mg, 0.27 mmol) and compound 11-3 (131 mg, 0.33 mmol) dissolved in N,N-dimethylformamide (3 mL) To 0 ° C, N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinium hexafluorophosphate (281 mg, 0.54 mmol) were added sequentially. After the addition, the reaction system was stirred at room temperature for 3 h. The liquid phase was purified to give the title compound 210 mg. ESI-MS (m/z): 1358.2 [M] ⁺ .

Step four:

4-carboxy-1-(4-(((S)-1-((())((()))) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl)-N,3-dimethyl Butyramide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido) Phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl Synthesis of quinine-1-anthracene bromide (compound 11-5)

To a mixed solvent of tetrahydrofuran (5 mL) and water (5 mL), EtOAc. A hydrochloric acid solution (0.5 mol/L) was added to adjust the pH = 3 to 4, and a part of the solvent was evaporated under reduced pressure to give a purified liquid. ESI-MS (m/z): 1330.2 [M] ⁺ .

Step five:

Synthesis of Compound 11

Compound 11-5 (58 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (2 mL). Carbonyl diimide hydrochloride (11 mg, 0.06 mmol) and 4-dimethylaminopyridine (15 mg, 0.12 mmol) were cooled to 0 ° C, and 2,3-difluorophenol (52 mg, 0.4 mmol) was added thereto. The temperature was raised to room temperature and allowed to react overnight. Purification by preparative liquid phase gave 4 mg of the title compound. ESI-MS (m/z): 1442.2 [M] ⁺ .

Example 12

4-((2,3-difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1,4-dimethylpiperidine-1-oxime iodide (Compound 12)

step one:

Synthesis of Ethyl 1-(4-(tert-Butoxy)-4-oxobutyl)-4-methylpiperidine-4-carboxylate (Compound 12-2)

Substituting compound 12-1 for compound 1-1 to give the title compound 130 mg. ESI-MS (m/z): 314.2 [M+H] ⁺ .

Step two:

1-(4-(tert-Butoxy)-4-oxobutyl)-4-(ethoxycarbonyl)-1,4-dimethylpiperidine-1-indole iodide (compound 12-3) synthesis

Substituting the compound 12-2 for the compound 1-2 to give the title compound. ESI-MS (m/z): 328.2 [M] ⁺ .

Step three:

Synthesis of 1-(3-carboxypropyl)-4-(ethoxycarbonyl)-1,4-dimethylpiperidine-1-oxime iodide (Compound 12-4)

A similar operation to that described in the third step of Example 1 was carried out, and Compound 1-3 was used instead of Compound 1-3 to give the title compound 100 mg. ESI-MS (m/z): 270.2 [M] ⁺ .

Step four:

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-4- Synthesis of (ethoxycarbonyl)-1,4-dimethylpiperidine-1-oxime iodide (Compound 12-5)

Substituting compound 12-4 for compound 1-4 to give the title compound 66 mg. ESI-MS (m/z): 1360.2 [M] ⁺ .

Step five:

4-carboxy-1-(4-(((S)-1-((())((()))) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl)-N,3-dimethyl Butyramide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido) Phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl Synthesis of 1-1,4-dimethylpiperidine-1-oxime iodide (compound 12-6)

Substituting compound 12-5 for compound 1-5 to give the title compound 50 mg. ESI-MS (m/z): 1332.2 [M] ⁺ .

Step six:

Synthesis of Compound 12

A similar operation as described in the first step of Example 1 was carried out, substituting compound 12-6 for compound 1-6 and 2,3-difluorophenol for 2,3,5,6-tetrafluorophenol to give the title compound 5 mg. ESI-MS (m/z): 1444.2 [M] ⁺ .

Example 13

4-((2,4-difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidin-1-indole iodide (Compound 14)

Example 14

4-((3,5-Difluorophenoxy)carbonyl)-1-(4-(((S)-1-((()))) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-) Butyramido)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Amido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutyl- Synthesis of 2-yl)amino)-4-oxobutyl)-1-methylpiperidine-1-oxime iodide (Compound 15)

A similar operation as described in the first step of Example 1 was carried out, and 2,5,5,6-tetrafluorophenol was replaced with 3,5-difluorophenol to give the title compound 5 mg. ESI-MS (m/z): 1430.2 [M] ⁺ .

Example 15

1-(4-(((S)-1-((())((()))) (3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-methylbutyryl)-N,3-dimethylbutyrylamide) 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl Amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-1- Synthesis of methyl-4-((3,4,5-trifluorophenoxy)carbonyl)piperidine-1-oxime iodide (Compound 20)

The title compound was obtained by substituting 3,4,5-trifluorophenol for 2,3,5,6-tetrafluorophenol using a procedure similar to that described in the procedure ESI-MS (m/z): 1448.2 [M] ⁺ .

Preparation of conjugate

Example 16. Coupling of Compound 2 with Trastuzumab

Under stirring, add 6 times the amount of compound 2 dissolved in N,N-dimethylacetamide (DMA) to 1 mL of trastuzumab solution at pH 7.5 and a concentration of 18 mg/ml. The coupling reaction was carried out for 4 h. Enrichment of different peak time components by HIC-HPLC gave specific conjugates with drug/antibody ratio (DAR) of 1 and 2, named 2-H1-1 and 2-H1-, respectively. 2.

Example 17. Coupling of Compound 7 with Trastuzumab

Using a coupling method similar to that of Example 16, compound 2 was replaced with compound 7, and the components of different peak times were enriched by HIC-HPLC to obtain specific conjugates having DAR values of 1 and 2, respectively designated as 1-H1-1 and 1-H1-2.

Example 18. Coupling of Compound 8 with Trastuzumab

Using a coupling method similar to that of Example 16, Compound 2 was replaced with Compound 8, and the components of different peak times were enriched by HIC-HPLC to obtain specific conjugates having DAR values of 1 and 2, respectively named 1-H1-1 and 1-H1-2.

Example 19. Coupling of Compound 9 with Trastuzumab

Using a coupling method similar to that of Example 16, compound 2 was replaced with compound 9, and the components of different peak times were enriched by HIC-HPLC to obtain specific conjugates having DAR values of 1 and 2, respectively designated as 1-H1-1 and 1-H1-2.

Example 20. Coupling of Compound 10 with Trastuzumab

Using a coupling method similar to that of Example 16, compound 2 was replaced with compound 10, and the components of different peak times were enriched by HIC-HPLC to obtain specific conjugates having DAR values of 1 and 2, respectively designated as 1-H1-1 and 1-H1-2.

Example 21. Coupling of Compound 11 with Trastuzumab

Using a coupling method similar to that of Example 16, compound 2 was replaced with compound 11, and the components of different peak times were enriched by HIC-HPLC to obtain specific conjugates having DAR values of 1 and 2, respectively named 11-H1-1 and 11-H1-2.

Example 22. Analysis of Conjugates by Hydrophobic Interaction Chromatography (HIC)

The reaction was monitored by HIC-HPLC and the conjugate was subjected to HIC detection.

The HIC conditions are as follows:

Liquid chromatography column: TOSOH TSKgel Butyl-NPR, 4.6x100mm

Mobile phase A: 1.5M ammonium sulfate

Mobile phase B: 25 mM Na ₂ HPO ₄ , pH 7.0, 25% isopropanol

Flow rate: 0.5ml/min

Detection wavelength: 280nm

Column temperature: 30 ° C

Sample chamber temperature: 8 ° C

Elution conditions:

时间(分钟)Time (minutes)	00	33	2525	3030	30.130.1	3535
流动相A(体积％)Mobile phase A (% by volume)	7070	7070	5555	1515	7070	7070
流动相B(体积％)Mobile phase B (% by volume)	3030	3030	4545	8585	3030	3030

The samples after the coupling of the naked anti-antibody and the

compounds

2, 7, 8, 9, 10 and 11 with the antibody were subjected to HIC-HPLC analysis, and the obtained spectra are shown in Figs. As can be seen from Figure 1, the bare anti-retention time is about 8.00 min. The overall analysis of Figure 2-7 shows that there is less residual resistance in the coupled system, and multiple conjugate peaks appear after the naked anti-peak, which proves that the bioactive molecule is highly coupled with the antibody.

Example 23. Determination of molecular weight

The coupled sample was subjected to LCMS molecular weight analysis.

Determination conditions:

Liquid chromatography column:

Protein BEH C4 1.7μm 2.1mm x 100mm

Mobile phase A: 0.1% formic acid (FA) / 98% H ₂ O / 2% acetonitrile (ACN)

Mobile phase B: 0.1% FA/2% H ₂ O/98% ACN

Flow rate: 0.25ml/min

Sample chamber temperature: 8 ° C

时间(分钟)Time (minutes)	11	77	88	99	1313
流动相A(体积％)Mobile phase A (% by volume)	9090	2020	2020	9090	9090
流动相B(体积％)Mobile phase B (% by volume)	1010	8080	8080	1010	1010

Mass Spectrometry: Triple TOF 5600+

GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10m/z 600-5000

The results are as follows: The theoretical molecular weights of the compound 2 and the compound 7 after coupling with the antibody are as follows:

糖型Glycoform	mAbmAb	DAR1DAR1	DAR2DAR2	DAR3DAR3	DAR4DAR4
G0F/G0FG0F/G0F	148057.8148057.8	149359.5149359.5	150661.1150661.1	151962.8151962.8	153264.5153264.5
G0F/G1FG0F/G1F	148220.0148220.0	149521.6149521.6	150823.3150823.3	152124.9152124.9	153426.6153426.6
G0F/G2FG0F/G2F	148382.1148382.1	149683.8149683.8	150985.4150985.4	152287.1152287.1	153588.7153588.7

In the table, mAb represents an antibody, DAR1 represents a conjugate comprising one biologically active molecule and one antibody, DAR2 represents a conjugate comprising two biologically active molecules and one antibody, and DAR3 represents a biologically active molecule and an antibody comprising three Conjugate, DAR4 represents a conjugate comprising four bioactive molecules and one antibody; glycoforms represent the sugar chain structure on two heavy chains, G0F represents fucosylation without galactose, and G1F represents fucosylated monogalactose G2F represents fucosylated digalactose.

The LCMS molecular weight analysis was carried out on the samples obtained by coupling the compound 2 and the compound 7 with the antibody, and the obtained spectrum is shown in Figs. 8 and 9. It can be seen from Fig. 8 and Fig. 9 that both compound 2 and compound 7 were successfully coupled with the antibody, and the coupled products were both DAR1 and DAR2.

Example 24. Coupling efficiency test

The coupling efficiency was calculated according to the analysis results of the conjugate by hydrophobic interaction chromatography (HIC), and the coupling efficiency of the

compounds

2, 7, 8, 9, 10 and 11 having a novel carboxyl activating group with the pentafluorophenol was used. The coupling efficiency of the control compound of the activated carboxyl group coupling technique with the antibody was compared, and the results are shown in Table 1. Wherein, the coupling efficiency refers to the molar percentage of the conjugate having the corresponding DAR value as a whole (all conjugates and bare reactance).

The structure of the control compound used in the coupling technique using a pentafluorophenol to activate a carboxyl group is as follows:

Table 1. Comparison of coupling efficiency

As is apparent from Table 1, the use of a compound having a novel carboxyl activating group in the present invention enables obtaining a conjugate of a biologically active molecule and one or two antibodies. The compound having a novel carboxyl activating group in the present invention significantly improves the coupling efficiency of the bioactive molecule and the antibody, and the multiple can be increased by 20 times or more, preferably 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more or 80 times or more, the problem that the bioactive molecule is difficult to couple with the antibody when the quaternary ammonium salt or the oxynitride structure is contained in the linker is successfully overcome.

Biological experiment

Example 25. Detection of antibody-drug conjugates inhibiting cell viability in vitro

The tumor cell HCC1954 was first cultured, and the medium was RPMI1640 + 10% FBS. HCC1954 is a Her2 positive cell with endocytosis to a conjugate (eg, an anti-Her2 antibody trastuzumab-drug conjugate). The conjugate was diluted with the corresponding detection medium (containing 2% FBS) (starting at 1 μg/mL, diluted 2 times, diluted 10 concentration gradients), and the tumor cells were digested by conventional methods using trypsin to collect tumor cells. Resuspend with the corresponding assay medium (containing 2% FBS). The diluted conjugate was added to a 96-well plate, and the resuspended cells were added to the corresponding wells containing the conjugate (10000 cells/well) and co-cultured for 3 days. Then, 20 μL of CCK8 reagent (Tongren Chemical Technology Co., Ltd.) was added to each well, and the reaction was carried out for 1.5 hours. The sample was read at 450 nm using a microplate reader (manufacturer: Molecular Devices, model: SpectraMax M2), and the dehydrogenase in the mitochondria was detected. Activity, evaluation of antibody-drug conjugate inhibition of cell proliferation.

The experimental results are shown in Table 2.

Table 2

ADC名称ADC name	EC ₅₀(ng/mL) EC ₅₀ (ng/mL)
1-H1-21-H1-2	13.48±0.4513.48±0.45

The conjugate of the present invention 1-H1-2 has an EC ₅₀ of 13.48 ± 0.45 ng / mL. The marketed Kadcyla (trastuzumab-maytansinoid T-DM1, trastuzumab-DM1 conjugate) has an EC ₅₀ of 43 ng/mL (see Howard A. Burris III et al., Clinical). Breast Cancer, Vol. 11, No. 5, 275-82). It can be seen that the conjugate of the present invention has a stronger inhibitory activity against cell proliferation than Kadcyla.

Although the invention has been illustrated by the foregoing specific embodiments, it should be understood that The present invention covers the general aspects of the invention, and various modifications and changes in the details of the invention may be made without departing from the spirit and scope of the invention. Accordingly, the description is for illustrative purposes only and not for purposes of limitation.

Claims

a method of preparing a conjugate of the formula (II),

The method comprises coupling a compound of formula (I) to a targeting drug comprising one or more amino groups (-NH 2 ),

among them:

Each occurrence of T is independently a group of a biologically active molecule, preferably a group that inhibits or interferes with the function of the cell and/or a biologically active molecule that causes cell death or destruction, and more preferably an antitumor biologically active molecule. Group

L 1 and L 2 are each independently a divalent linking group at each occurrence;

Each occurrence of L 3 is independently a cyclic or chain group comprising at least one quaternized nitrogen atom or a cyclic or chain group comprising at least one nitrogen atom substituted by =0;

Each occurrence of R 1 is independently a substituted or unsubstituted C 1-6 alkylene group, a substituted or unsubstituted C 2-10 alkenylene group, a substituted or unsubstituted C 2 . -10 alkynylene, substituted or unsubstituted C 3-10 cycloalkylene, substituted or unsubstituted C 6-10 arylene or substituted or unsubstituted 5-10 membered hetero Aryl;

Z is an oxygen atom or a sulfur atom, and is preferably an oxygen atom;

R 2 is selected from the group consisting of C 1-6 alkyl, C 3-10 cycloalkyl, 5-10 membered heterocyclyl, C 6-10 aryl and 5-10 substituted with one or more R a Metaheteroaryl;

R a is halogen (for example, fluorine, chlorine or bromine), cyano, nitro or trifluoromethyl, provided that when R a is fluorine, R 2 is not a perfluorophenyl group;

A is a group obtained by removing α amino groups in a targeted drug, and the targeted drug includes a macromolecule and a small molecule having a targeting effect, such as an antibody, DNA, RNA or a small molecule ligand;

m, n and r are each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 at each occurrence; preferably, m and n are each present at each occurrence Independently 0, 1, 2 or 3, and r is each independently 0, 1 or 2 at each occurrence; more preferably, m and n are 1 and r is 0;

α is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, α is 1, 2, 3 or 4; more preferably, α is 1 or 2.
The method of claim 1 wherein T is a group selected from the group consisting of a metal complex such as a metal platinum complex (e.g., oxaliplatin) or a metal gold complex; a glycopeptide antibiotic, for example Bleomycin or pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan) , topotecan, belocommide or rupibine) or topoisomerase II inhibitors (such as actinomycin D, doxorubicin, doxorubicin, doxymimethine, daunorubicin, rice Toxic, podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, guanidine, pentastatin, fludarabine, Cladribine or nairabine; drugs acting on structural proteins, such as tubulin inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel); Tumor signaling pathway inhibitors, such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, Aspartate kinase inhibitor or histidine kinase inhibitor; proteasome inhibitor; histone deacetylase inhibitor; tumor angiogenesis inhibitor; cyclin inhibitor; maytansine derivative; Pyridin derivatives; auristatin derivatives; pyrrolobenzodiazepine
Dimer (PBD) derivatives; melphalan; mitomycin C; and chlorambucil or other biologically active molecules that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis;

Preferably, T is selected from the following structures:

and

More preferably, T is selected from the following structures:
The method according to claim 1 or 2, wherein L 1 is selected from the group consisting of an amino acid group, a peptide group, an oligosaccharide group, -(CH 2 ) t -, -(CH 2 CH 2 O) t -(CH 2 ) t -, -(CH 2 ) t -(CH 2 CH 2 O) t -,

The group is linked to T by one of the two positions labeled 1 or 2 and to L 2 by another position; preferably, the group is linked to T by a position of 1 mark and is marked by 2 Position connected to L 2 ;

among them:

R j and R k are each independently selected from H (hydrogen), D (oxime), halogen, -COOH, -SO 3 H, CF 3 , CN, CH 2 CN, C 1-6 alkyl, C 1-6 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl and C 3-6 cycloalkyl;

t each independently is an integer of 1, 2, 3, 4, 5 or 6 at each occurrence;

x is an integer of 0, 1, 2, 3, 4, 5 or 6;

z is an integer of 0, 1, 2 or 3;

Preferably, L 1 is selected from the group consisting of a peptide group consisting of 2-5 amino acids, an oligosaccharide group,

The group is linked to T by one of the two positions labeled 1 or 2 and to L 2 by another position; preferably, the group is linked to T by a position of 1 mark and is marked by 2 The position is connected to L 2 ;

More preferably, L 1 is
The group is linked to T by one of the two positions labeled 1 or 2 and to L 2 by another position; preferably, the group is linked to T by a position of 1 mark and is marked by 2 The location is connected to L 2 .
The method according to any one of claims 1 to 3, wherein L 2 is a group selected from the group consisting of an amino acid group, a peptide group composed of 2 to 10 amino acids, an oligosaccharide group, -(CH 2 y -, -(CH 2 CH 2 O) y -(CH2) y -, -(CH 2 ) y -(CH 2 CH 2 O) y -,

The group is linked to L 1 by one of the two positions labeled 1 or 2 and to L 3 by another position; preferably, the group is linked to L 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 3 ;

among them:

R j , R k , R m and R n are each independently selected from H (hydrogen), D (oxime), halogen, -COOH, -SO 3 H, carboxylic acid, sulfonic acid, CF 3 , CN, CH 2 CN a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-10 alkenyl group, a C 2-10 alkynyl group, and a C 3-6 cycloalkyl group;

y each independently is an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

Preferably, L 2 is selected from the group consisting of an amino acid group, a peptide group consisting of 2-5 amino acids,

The group is linked to L 1 by one of the two positions labeled 1 or 2 and to L 3 by another position; preferably, the group is linked to L 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 3 ;

More preferably, L 2 is a group selected from the group consisting of:
The group is linked to L 1 by one of the two positions labeled 1 or 2 and to L 3 by another position; preferably, the group is linked to L 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 3 ; further preferably, L 2 is
The group is attached to L 1 through the position of the 1 mark and to the L 3 through the position of the 2 mark.
The method of any one of claims 1 to 4, wherein L 3 is selected from the group consisting of the following: optionally substituted by one or more R i :
a 3-8 membered nitrogen-containing heterocyclylene group containing at least one quaternized nitrogen atom, a 6-12 membered nitrogen-containing ylidene heterocyclic group, a 6-12 membered nitrogen-containing siroheterocyclyl group, and a 6-12 member a nitrogen-containing heterocyclic heterocyclic group, a nitrogen-containing heteroarylene group, a C 3-8 cycloalkylene group-W-, and a 3-8 membered nitrogen-containing heterocyclylene group containing at least one nitrogen atom substituted by =0, wherein W is
E is a counter ion, and each of R p is independently selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 alkoxy, and -C 1-2 alkyl-cyano at each occurrence. And R q is each independently selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and C 3-8 cycloalkyl at each occurrence;

Preferably, L 3 is selected from optionally substituted with one or more substituents R i of the following groups:
a 5-6 membered nitrogen-containing heterocyclic heterocyclic group containing at least one quaternized nitrogen atom, a 8-11 membered nitrogen-containing ylidene heterocyclic group, a 8-11 membered nitrogen-containing snail heterocyclic group, and 8-11 members a nitrogen-containing heterocyclic heterocyclic group, a 5-6 membered nitrogen-containing heteroarylene group, a C 5-6 cycloalkylene group-W-, and a 5-6 membered nitrogen-containing heteropoly group containing at least one nitrogen atom substituted by =O Ring base, where W is
E is a counter ion, and each of R p is independently selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 alkoxy, and -C 1-2 alkyl-cyano at each occurrence. And R q is each independently selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and C 3-8 cycloalkyl at each occurrence;

Preferably, L 3 is selected from optionally substituted with one or more substituents R i of the following groups:
a 5-6 membered nitrogen-containing heterocyclylene group containing at least one quaternized nitrogen atom, an 8 membered nitrogen-containing ylidene heterocyclic group, a 11-membered nitrogen-containing snail heterocyclic group, and a 8-11 membered nitrogen-containing snail a cyclic group, a 5-6 membered nitrogen-containing heteroarylene (eg, pyridylene), a C 5-6 cycloalkylene-W-, and a 5-6 membered nitrogen containing at least one nitrogen atom substituted with =O Heterocyclylene, wherein W is
E is a counter ion, and each of R p is independently selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 alkoxy, and -C 1-2 alkyl-cyano at each occurrence. And R q is each independently selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and C 3-8 cycloalkyl at each occurrence;

Preferably, L 3 is selected from optionally substituted with one or more substituents R i of the following groups:
a 5-6 membered nitrogen-containing heterocyclic heterocyclic group containing at least one quaternized nitrogen atom, an 8 membered nitrogen-containing ylidene heterocyclic group, an 11-membered nitrogen-containing snail heterocyclic group, and an 8-membered nitrogen-containing fused heterocyclic group a 6-membered nitrogen-containing heteroarylene group, a cyclohexylene group-W-, and a 5-6 membered nitrogen-containing heterocyclylene group containing at least one nitrogen atom substituted with =O, wherein W is
E is a counterion, each of R p is independently selected from -CH 2 CN and -CH 2 CH 2 CN, and R q is each independently selected from C 1-6 alkyl, C at each occurrence. 2-6 alkenyl, C 2-6 alkynyl and C 3-8 cycloalkyl;

More preferably, L 3 is selected from the group consisting of the following: optionally substituted by one or more R i :

The group is attached to R 1 by one of two positions labeled 1 or 2 and to L 2 via another position; preferably, the group is linked to R 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 2 ;

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion;

R q is each independently selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and C 3-8 cycloalkyl at each occurrence;

R p is each independently selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 alkoxy and -C 1-2 alkyl-cyano, preferably -CH, in each occurrence. 2 CN and -CH 2 CH 2 CN;

β is an integer of 0, 1, or 2;

γ is each independently an integer of 1, 2 or 3 at each occurrence;

R i is selected from the group consisting of H (hydrogen), D (oxime), halogen, =O, CF 3 , CN, CH 2 CN, C 1-6 alkyl, C 1-6 alkoxy, C 2-10 alkenyl, C 3-8 cycloalkyl, C 2-10 alkynyl, COOH and SO 3 H, preferably, R i is selected from H (hydrogen), D (氘), =O, CN, CH 2 CN, methyl and CF 3 ;

More preferably, L 3 is selected from the group consisting of the following: optionally substituted by one or more R i :

The group is attached to R 1 by one of two positions labeled 1 or 2 and to L 2 via another position; preferably, the group is linked to R 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 2 ;

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion;

R q is each independently selected from C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl and C 3-6 cycloalkyl at each occurrence;

R i is selected from the group consisting of H (hydrogen), D (氘), CN, CH 2 CN, methyl and CF 3 ;

More preferably, L 3 is selected from the group consisting of:

The group is attached to R 1 by one of two positions labeled 1 or 2 and to L 2 via another position; preferably, the group is linked to R 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 2 ;

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion;

R q is each independently selected from the group consisting of C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, and C 3-6 cycloalkyl at each occurrence; and R i is selected from H ( Hydrogen), D(氘), CN, CH 2 CN, methyl and CF 3 ;

Further preferably, L 3 is selected from the group consisting of

The group is attached to R 1 by one of two positions labeled 1 or 2 and to L 2 via another position; preferably, the group is linked to R 1 by the position of 1 mark, and passes through 2 The position of the mark is connected to L 2 ;

among them:

E is a counter ion, preferably a halogen anion, and more preferably a chloride ion, a bromide ion or an iodide ion.
The method of any one of claims 1 to 5, wherein R 1 is selected from the group consisting of: C 1-6 alkylene, C 3-10 subring, optionally substituted by one or more R b An alkyl group, a C 6-10 arylene group and a 5-10 membered heteroarylene group, wherein R b is H (hydrogen), D (氘), halogen, cyano, nitro, trifluoromethyl, COOH or SO 3 H.
The method of any of claims 1-6, wherein
From:

The group is attached to L 1 through the position of 1 mark, and is attached to the carbonyl group through the position of 2 mark;

Preferably,
From:

The group is attached to L 1 through the position of the 1 tag and is attached to the carbonyl group via the position of the 2 tag.
The method according to any one of claims 1 to 7, wherein R 2 is selected from the group consisting of: C 1-6 alkyl, C 3-8 cycloalkyl optionally substituted by one or more R a , 5-10 yuan heteroaryl,
And R a is fluorine, chlorine, bromine, nitro or trifluoromethyl, provided that when R a is halogen, R 2 is not a perhalogenated C 6-10 aryl;

Preferably, R 2 is selected from the group consisting of optionally substituted by one or more R a : C 1-6 alkyl, 5-10 membered heteroaryl,
And R a is fluorine, nitro or trifluoromethyl, provided that when R a is fluorine, R 2 is not a perfluoro C 6-10 aryl;

More preferably, R 2 is selected from the group consisting of: C 1-6 alkyl and optionally substituted by one or more R a
And R a is fluorine or a nitro group, provided that when R a is fluorine, R 2 is not a perfluorophenyl group;

More preferably, R 2 is a phenyl group substituted with four fluorine atoms, a phenyl group substituted with three fluorine atoms, a phenyl group substituted with two fluorine atoms, a phenyl group substituted with a fluorine atom, a phenyl group substituted with a nitro group, and a hexene group. a fluorine atom-substituted C 3-6 alkyl group, five fluorine atom-substituted C 2-6 alkyl groups, four fluorine atom-substituted C 2-6 alkyl groups or three fluorine atom-substituted C 1-6 alkyl groups ;

More preferably, R 2 is selected from the group consisting of

and

Further preferably, R 2 is 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl or 2,6-difluorophenyl.
The method of any of claims 1-8, wherein
From:

Preferably,
From:

More preferably,
From:

and

Further preferably,
From:
The method according to any one of claims 1 to 9, wherein the targeted drug is selected from the group consisting of: small molecule ligands, proteins, polypeptides, antibodies, and non-protein drugs such as sugars, RNA or DNA;

Preferably, the targeted drug is selected from the group consisting of: a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (such as a carbonic anhydrase IX inhibitor). ; RGD peptides that recognize cell surface integrin receptors; growth factors that recognize cell surface growth factor receptors such as EGF, PDGF or VEGF; recognize functional cell surface plasminogen activator, bombesin, bradykinin , somatostatin or peptide of prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, DR5 ligand, linkage a two aliphatic steroid polyene, a cyanine dye, IR-783 or a derivative thereof; and an antibody or an active fragment or variant thereof, such as a monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or Antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragments, single chain antibodies (eg, scFv), non-human antibodies, humanized antibodies, chimeric antibodies, all Human antibody, pro-antibody, bispecific antibody or multispecific antibody;

Preferably, the targeted drug is a targeted drug targeting the following targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEACAM5, mucin (eg sticky Protein 1 and mucin 16), CD138, CD20, CD19, CD30, SLTRK6, connexin 4, tissue factor, endothelin receptor, STEAP1, SLC39A6, guanylate cyclase C, PSMA, CCD79b, CD22, sodium phosphate Co-transporter 2B, GPNMB, trophoblastic glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783 , STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, short proteoglycan, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, sealing protein 18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD 45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1 AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2 , IGF-1R, RNF43 and NaPi2b;

More preferably, the targeting drug is a monoclonal antibody against Her2 or an active fragment or variant thereof, or a monoclonal antibody against Trop-2 or an active fragment or variant thereof;

Further preferably, the targeted drug is trastuzumab, pertuzumab or Sacituzumab.
The method according to any one of claims 1 to 10, wherein the compound of the formula (I) is selected from the group consisting of:
A method according to any one of claims 1 to 11 comprising mixing a targeted drug with a compound of formula (I);

Preferably, the molar ratio of the targeted drug to the compound of formula (I) is 1: (1-20);

Preferably, the method comprises mixing a solution comprising a targeted drug with a compound of formula (I);

Preferably, the method is carried out in water or an organic solvent;

Preferably, the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as Methylene chloride, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols ( Such as methanol or ethanol) and any combination thereof;

Preferably, the method further comprises purifying by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, and affinity chromatography.
a conjugate of the formula (II), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, wherein the conjugate of the formula (II) is as claimed in claim 1 Prepared by the method of any of 12,

Wherein each group is as defined in any one of claims 1-12;

Preferably, the conjugate of the formula (II) is an antibody-drug conjugate;

Preferably, the antibody-drug conjugate is:

Wherein a is an integer of 1, 2, 3 or 4; and A is a group obtained by removing α amino groups from trastuzumab, pertuzumab or Sacituzumab;

Preferably, the antibody-drug conjugate is:

Wherein A1 is a group obtained by removing two amino groups from trastuzumab;

Preferably, the antibody-drug conjugate is:

Wherein A2 is a group obtained by removing two amino groups in pertuzumab;

Preferably, the antibody-drug conjugate is:

Wherein A3 is a group obtained by removing two amino groups in Sacituzumab;

Preferably, the antibody-drug conjugate is:

Wherein A1' is a group obtained by removing one amino group from trastuzumab;

Preferably, the antibody-drug conjugate is:

Wherein A2' is a group obtained by removing one amino group from pertuzumab; and

Preferably, the antibody-drug conjugate is:

Wherein A3' is a group obtained by removing one amino group from Sacituzumab.
Process for the preparation of a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, which comprises reacting a compound of formula (I-0) with R 2- ZH reaction to give a compound of the formula (I),

Wherein each group is as defined in any one of claims 1-12.
a compound of the formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,

Wherein each group is as defined in any one of claims 1-12;

Preferably, the compound of formula (I) is prepared by the method of claim 14;

Preferably, the compound of the formula (I) has the structure of the formula (Ia),

More preferably, the compound is selected from the group consisting of
A pharmaceutical composition comprising a conjugate prepared by the method of any one of claims 1 to 12, a conjugate of claim 13 or a pharmaceutically acceptable salt of the conjugate, a stereo Isomers or metabolites or solvates thereof and one or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more other anticancer agents such as chemotherapeutic agents and/or Or antibody.
A pharmaceutical preparation comprising a conjugate prepared by the method according to any one of claims 1 to 12, a conjugate according to claim 13, a pharmaceutically acceptable salt of the conjugate, a stereo Isomer or metabolite or solvate thereof, or the pharmaceutical composition of claim 16.
A conjugate prepared by the method of any one of claims 1 to 12, a conjugate of claim 13, a pharmaceutically acceptable salt, stereoisomer or metabolite of the conjugate Or the use of the solvate thereof, the pharmaceutical composition according to claim 16, or the pharmaceutical preparation according to claim 17 for the preparation of a medicament for preventing or treating a cancer disease;

Preferably, the cancer disease is selected from the group consisting of esophageal cancer (eg, esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (eg, small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer , gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, salivary gland cancer, colon cancer, colorectal cancer, liver cancer, kidney cancer, solid tumor, non-Hodgkin's lymphoma , central nervous system tumors (such as glioma, glioblastoma multiforme or glioma or sarcoma), prostate cancer and thyroid cancer.
A conjugate prepared by the method of any one of claims 1 to 12, a conjugate of claim 13, a pharmaceutically acceptable salt, stereoisomer or metabolite of the conjugate Or a solvate thereof, the pharmaceutical composition according to claim 16, or the pharmaceutical preparation according to claim 17 for preventing or treating a cancer disease;

Preferably, the cancer disease is selected from the group consisting of esophageal cancer (eg, esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (eg, small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer , gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, salivary gland cancer, colon cancer, colorectal cancer, liver cancer, kidney cancer, solid tumor, non-Hodgkin's lymphoma , central nervous system tumors (such as glioma, glioblastoma multiforme or glioma or sarcoma), prostate cancer and thyroid cancer.
A method of preventing or treating a cancer disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate prepared by the method according to any one of claims 1 to 12, the coupling of claim 13. And a pharmaceutically acceptable salt, stereoisomer or metabolite of the conjugate or a solvate thereof, the pharmaceutical composition according to claim 16, or the pharmaceutical preparation according to claim 17;

Preferably, the cancer disease is selected from the group consisting of esophageal cancer (eg, esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (eg, small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer , gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, salivary gland cancer, colon cancer, colorectal cancer, liver cancer, kidney cancer, solid tumor, non-Hodgkin's lymphoma , central nervous system tumors (such as glioma, glioblastoma multiforme or glioma or sarcoma), prostate cancer and thyroid cancer.