Camptothecin derivatives and their ligand-drug conjugates

Abstract

The present invention discloses camptothecin derivatives and their ligand-drug conjugates, which provide superior antitumor camptothecin-based ADC drugs with higher safety and efficacy, and can better meet clinical needs.

Description

[Technical Field]

[0001] This invention relates to camptothecin derivatives and their ligand-drug conjugates. [Background technology]

[0002] As a new form of targeted therapy, ligand-drug conjugates (ADCs) typically consist of three parts: an antibody or antibody-based ligand, a small molecule drug, and a binding unit that connects the ligand and the drug. Antibody-drug conjugates utilize the specific recognition of an antibody against an antigen to deliver the drug molecule to target cells, effectively releasing the drug molecule and achieving therapeutic objectives. In August 2011, the U.S. Food and Drug Administration (FDA) approved AdcetrIs, a new ADC drug developed by Seattle Genetic Ics for the treatment of Hodgkin lymphoma and relapsing large cell lymphoma (ALCL). TM The drug has been approved for market release. Its safety and efficacy have been demonstrated through clinical use.

[0003] Camptothecines (including irinotecan, exatecan, and SN38), small molecule compounds with antitumor properties, exert their antitumor effects by inhibiting DNA topoisomerase I. Many camptothecin drugs are widely used in clinical practice, with their main indications being bone cancer, prostate cancer, breast cancer, and pancreatic cancer. Unlike irinotecan, which is currently in clinical use, exatecan does not require enzymatic activation. Furthermore, compared to SN-38, the active ingredient in irinotecan, and topothecan, which is used clinically, exatecan has stronger inhibitory activity against topoisomerase I, causing stronger damage to various cancer cells in vitro. In particular, the expression of P-glycoprotein has shown efficacy even in cancer cells resistant to SN-38 and other agents. Exatecan has not been successfully marketed as a monotherapy drug, and it is speculated that its high cellular activity limits its therapeutic range.

[0004] The advantages of antibody-drug conjugates (ADCs) lie in increased water solubility, improved targeting, specific binding to antigens, drug transport to the vicinity of target cells, tumor cell death through drug release near target cells, and reduced toxic side effects. Camptothecin-based drugs show considerable potential in the field of ADCs. Currently, trastuzumab deruxtecan (trade name: Enhertu), an antibody-conjugated drug with exatecan as its toxin, was approved for market by the US FDA on December 20, 2019. As the first commercially available camptothecin-based ADC, it has fully demonstrated the drug discovery capabilities and application prospects of such drugs in the ADC field.

[0005] Enhertu is an ADC drug targeting HER2, with approximately eight exatecan derivatives (DAR=8) conjugated to a single antibody. However, Daiichi Sankyo, the research and development company, recognized the need to reduce the DAR value for safety reasons. Daiichi Sankyo's clinically tested ADC, DS-1062, has a reduced DAR value of 4. A reduction in the DAR value may decrease therapeutic efficacy. Therefore, there is a need to develop safer camptothecin derivatives.

[0006] In ADC drugs, the drug's DAR value is reduced to ensure safety. As a result, the therapeutic effect is diminished. Drug solubility is one of the factors that affect the DAR value and is also a factor that affects the bystander effect of ADC drugs. Camptothecin-based drugs are widely used clinically, but their low water solubility remains a problem that needs to be addressed. For drugs with low water solubility, improving the drug's water solubility can improve its physical properties and metabolic status in the body, thereby improving pharmacological efficacy, enhancing therapeutic effects, reducing drug dosage, and lowering costs. Low water solubility increases the degree of aggregation of ADCs, making them more susceptible to endocytosis by normal cells, leading to toxicity and side effects, or they are more easily metabolized and have a shorter half-life.

[0007] During our research, we unexpectedly discovered that the amino enantiomer compounds of exatecan have significantly better water solubility than exatecan itself. The derivatives synthesized using this method also exhibit superior water solubility, enabling the production of superior antitumor camptothecin-based ADC drugs with higher safety and efficacy, better meeting clinical needs. [Overview of the Initiative]

[0008] The present invention provides camptothecin derivatives represented by general formula D, tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof. [ka] Here, [ka] The chiral carbon atom bonded to it has an R absolute configuration. R is selected from hydrogen atoms, deuterium atoms, halogens, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl. R1 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R2 This is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. X is -C(O)-CR a R b -(CR3R4) m -O-, -C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CRa R b -(CR3R4)- m is selected from -S- and, R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a deuterated alkyl, a halogenated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclyl, an aryl, a substituted aryl or a heteroaryl, R b is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a deuterated alkyl, a halogenated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclyl, an aryl, a substituted aryl or a heteroaryl, or R a R b and the carbon atom to which they are attached form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl, R3 and R4 may be the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a halogenated alkyl, a deuterated alkyl, an alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyalkyl, a cycloalkyl or a heterocyclyl, or R3, R4 and the carbon atom to which they are attached form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl, m is selected from integers from 0 - 4.

[0009] Preferably, R1 is selected from C 1-3 alkyl.

[0010] Preferably, R2 is selected from C 1-3 alkyl or a fluorine atom.

[0011] The camptothecin derivative has a structure represented by the following formula D1.

Chemical Formula

[0012] Preferably, R is C 1-3 Selected from alkyl groups.

[0013] Preferably, X is not limited to [ka] Selected from the above, where the wavy line on the left is bound to the camptothecin derivative moiety, and the wavy line on the right is bound to the binding unit.

[0014] Preferably, the compound is not limited to [ka] R is selected from, where R is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a substituted alkyl, a deuterated alkyl, a cycloalkylalkyl, an alkoxyalkyl, an aryl, a substituted aryl, or a heteroaryl.

[0015] More preferably, the compound is not limited to [ka] Selected from.

[0016] The present invention provides linker-drug conjugates containing camptothecin derivatives, or pharmaceutically acceptable salts or solvates thereof, tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof. [ka] Here, [ka] The chiral carbon atom bonded to it has an R absolute configuration. R is a hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl; R1 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R2 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. X is -C(O)-CR a R b -(CR3R4) m -O-, -C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CR a R b -(CR3R4) m Selected from -S-, R a This is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R b is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls, or R a , R b and the carbon atoms bonded to them are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, R3 and R4 may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, alkyl halide, alkyl deuterated, alkoxy, hydroxyl, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclyl, or R3, R4, and the carbon atoms bonded to them are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, m is selected from integers between 0 and 4. L is -L1-L2-L3-L4-.

[0017] Preferably, -L- is -L1-L2-L3-L4-, where the L1 end is bound to ligand Ab and the L4 end is bound to X.

[0018] More preferably, L1 is not restricted. [ka] Selected from.

[0019] More preferably, L2 is -NC(R5R6)C(O), -NR7(CH2) o C(O)-, -NR7(CH2CH2O) o CH2C(O)-, -S(CH2) p It is selected from C(O)- or chemical bonds, where o is selected from integers between 0 and 20, and p is selected from integers between 0 and 20. R5 and R6 may be the same or different, and each is independently selected from hydrogen, deuterium, alkyl, substituted alkyl, deuterated alkyl, heteroalkyl, carboxyl, amino, and substituted amino. R7 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, aryls, substituted aryls, or heteroaryls. L1 and L2 share a nitrogen atom.

[0020] More preferably, L3 is selected from peptide residues consisting of amino acids. Selectively, the amino acid is further substituted with one or more substituents selected from deuterium atoms, halogens, hydroxyl, cyano, amino, nitro, carboxyl, alkyl, substituted alkyl, alkoxy and cycloalkyl or substituted cycloalkyl, and is preferably selected from peptide residues formed from one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D).

[0021] More preferably, L4 is -NR8(CR9R 10 ) q -, -C(O)NR8-, -C(O)NR8(CH2) q -or selected from chemical bonds, where q is an integer selected from 0-6. R8, R9 and R 10 These elements may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls.

[0022] Preferably, the coupling unit L- is not limited to [ka] Selected from JPEG0007860665000012.jpg237113, JPEG0007860665000013.jpg22288, and JPEG0007860665000014.jpg16595, where the wavy line on the left is bound to the ligand portion and the wavy line on the right is bound to X.

[0023] More preferably, the linker-drug complex is not limited to [ka] Selected from JPEG0007860665000016.jpg211114JPEG0007860665000017.jpg23185JPEG0007860665000018.jpg13283, where the carbon atom at position 1 has either an R absolute configuration or an S absolute configuration.

[0024] The present invention further provides ligand-drug complexes comprising a linker-drug complex, and pharmaceutically acceptable salts or solvates thereof. The ligand-drug complex has a structure represented by formula I. [ka] Formula I Here, [ka] The chiral carbon atom bonded to it has an R absolute configuration. R is selected from hydrogen atoms, deuterium atoms, halogens, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl. R1 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R2 is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, substituted alkyls, deuterated alkyls, cycloalkylalkyls, alkoxyalkyls, carboxyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. X is -C(O)-CR a R b -(CR3R4) m -O-, -C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CR a R b -(CR3R4) m Selected from -S-, R aThis is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R b is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls, or R a , R b and the carbon atoms bonded to them are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, R3 and R4 may be the same or different, and each may independently be a hydrogen atom, a deuterium atom, a halogen, an alkyl, an alkyl halide, an alkyl deuterated, an alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyalkyl, a cycloalkyl, or a heterocyclyl. R3, R4, and the carbon atoms bonded to them are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, Ab is a ligand unit, selected from antibodies, antibody fragments, target proteins, Fc-fusion proteins, etc. L is the unit that binds to Ab, and X is the drug partial modification unit. m is selected from integers between 0 and 4, and n is selected from integers or decimals between 1 and 20.

[0025] Preferably, the ligand is selected from mouse antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0026] More preferably, the antibody or its antigen-binding fragment is selected from, without limitation, anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody.

[0027] More preferably, the ligand-drug conjugate is, without limitation [Chemical formula] Selected from JPEG0007860665000022.jpg226148, JPEG0007860665000023.jpg251127, and JPEG0007860665000024.jpg52119. Here, Ab is a ligand unit, n is an integer or decimal selected from 1 to 20, and the 1-position carbon atom has either an R absolute configuration or an S absolute configuration.

[0028] The present invention further provides a method for producing linker-drug conjugates, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof. This method includes the following steps: [ka] Connecting Unit L a And, through a substitution reaction with a compound represented by general formula D1, general formula L a -Obtain a linker-drug complex represented by X-D1 Here, D 1 of [ka] The chiral carbon atoms bonded to them have an R absolute configuration, L2, L3, R, R8, R9, R 10 q and X are the same as those in the general formula LXD.

[0029] The present invention further provides a method for producing ligand-drug conjugates, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, comprising the following steps. [ka] A ligand-drug conjugate represented by the general formula Ab-LX-D1 is obtained by a coupling reaction between a reduced antibody, antibody fragment, or its antigen-binding fragment and the general formula (LX-D1). Here, [ka] The chiral carbon atom bonded to it has an R absolute configuration, and Ab, L, X, R, and n are the same as in general formula I.

[0030] Camptothecin derivatives, linker-drug conjugates, ligand-drug conjugates, or pharmaceutically acceptable salts or solvates thereof, wherein the pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts formed with acidic functional groups in the structural formula; or acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbicates, salicylates, formates, glutamates, mesylates, ethanesulfons, benzenesulfons or p-toluenesulfons formed with basic functional groups in the structure.

[0031] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the camptothecin derivative, a linker-drug conjugate, a ligand-drug conjugate, a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0032] The present invention provides for the use of camptothecin derivatives, linker-drug conjugates, ligand-drug conjugates, tautomers, meso compounds, racemic compounds, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts or solvent compounds thereof, and pharmaceutically acceptable carriers, diluents or excipients in the manufacture of drugs for treating or preventing tumors.

[0033] Preferably, the tumor is a solid tumor or hematological malignancy such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, or leukemia. [Modes for carrying out the invention]

[0034] Detailed description of the invention Unless otherwise defined, all technical and scientific terms used herein are the same as those commonly understood by an ordinary person skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure, but exemplary methods and materials are described herein. In describing and asserting the invention, the following terms are used according to the following definitions.

[0035] When a product name is used in this specification, it is intended to include the formulation, generic drug, and active ingredient of the product to which the product name pertains.

[0036] Unless otherwise stated, terms used herein and in the claims have the same meanings as set forth below.

[0037] The term "ligand" refers to a macromolecule that can recognize and bind to an antigen or receptor associated with a target cell. The role of a ligand is to present a drug to the target cell population to which the ligand binds. Such ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In embodiments of the present invention, the ligand is represented by Ab. The ligand can be bound to a binding unit via a heteroatom on the ligand. Preferably, it is an antibody or an antigen-binding fragment thereof. The antibody is selected from chimeric antibodies, humanized antibodies, fully human antibodies, and mouse antibodies, and is preferably a monoclonal antibody.

[0038] A ligand unit is a targeting agent that specifically binds to a target site. Ligands can specifically bind to cellular components or to target molecules of cellular components or other eyes. The target site or target is typically on the cell surface. In some embodiments, the function of the ligand unit is to deliver the drug unit to a specific population of target cells that interact with the ligand unit. Ligands include, but are not limited to, proteins, polypeptides, peptides, and non-protein molecules such as sugars. Suitable ligand units include antibodies, such as full-length (intact) antibodies and their antigen-binding fragments. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the binding unit is covalently bonded to the sulfur atom of the ligand. In some embodiments, the sulfur atom is the sulfur atom of a cysteine ​​residue, forming an interchain disulfide bond of the antibody. In another embodiment, the sulfur atom is the sulfur atom of a cysteine ​​residue to which the ligand unit has been introduced, forming an interchain disulfide bond of the antibody. In yet another embodiment, the sulfur atom is the sulfur atom of a cysteine ​​residue to which the ligand unit has been introduced (e.g., by site-directed mutagenesis or chemical reaction). In yet another embodiment, the sulfur atom bound to the binding unit is selected from the cysteine ​​residue of the interchain disulfide bond of the antibody and the cysteine ​​residue to which the ligand unit has been introduced (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the EU index numbering system described in Kabat EA et al., (1991), Sequences of proteIns of Immunologic Interest, Fifth Edition, NIH publication 91-3242.2 is used.

[0039] In this specification, the terms “antibody” or “antibody unit” include, to the extent that they apply, any part of an antibody structure. This unit can bind to, reactively interact with, or form complexes with receptors, antigens, or other receptor units possessed by a target cell population. The antibody may be any protein or protein-like molecule that can bind to, form complexes with, or react with a portion of a cell population being treated or biologically modified. In the present invention, the antibody constituting the antibody-drug conjugate may maintain its original wild-state antigen-binding ability. Therefore, the antibody of the present invention may preferably bind specifically to an antigen. Such antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (with known or predictable functionality), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (with known or predictable functionality). As described herein, tumor-associated factors may also be cluster differentiation factors (such as CD proteins).

[0040] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for antibody preparation. To develop effective cellular-level targets for cancer diagnosis and treatment, researchers are finding transmembrane or other tumor-associated polypeptides. These targets are little to no expression on the surface of one or more non-cancer cells but can be specifically expressed on the surface of one or more cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells compared to the surface of non-cancer cells. Identifying such tumor-associated factors can significantly improve the specific targeting characteristics of antibody-based cancer treatment. For convenience, antigen-related information (names, other designations, GenBank accession numbers, etc.) that is well known in the art is listed below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as GenBank. The tumor-associated antigens corresponding to the antibody targets include all amino acid sequence variants and isotypes, and have at least 70%, 80%, 85%, 90%, or 95% identity with the sequences identified in the references, or possess biological properties and characteristics that are completely identical to the sequences of tumor-associated antigens described in the references.

[0041] The terms "inhibit" or "suppress" refer to reducing a detectable amount or preventing it completely.

[0042] The term "cancer" refers to a physiological condition or disease characterized by uncontrolled cell proliferation. "Tumor" includes cancer cells.

[0043] The term "autoimmune disease" refers to a disease or disorder caused by the targeting of an individual's own tissues or proteins.

[0044] The term "drug" refers to cytotoxic drugs, which may be indicated as D, and are chemical molecules that can strongly inhibit the normal growth of tumor cells. While cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations, they lack specificity and, while killing tumor cells, can also cause apoptosis in normal cells, potentially leading to serious side effects. This term also includes toxins such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 BI 212 , P 32 The substance may include radioactive isotopes of Lu176, toxic drugs, chemotherapeutic agents, antibiotics, and ribolicin, but it is preferable that the substance be a toxic drug.

[0045] The terms “binding unit,” “binding unit fragment,” or “binding unit” refer to a chemical structural fragment or bond that has one end bound to a ligand and the other end bound to a drug, which may bind to another binding unit before binding to the drug.

[0046] The binding unit comprises a stretcher, a spacer, and an amino acid unit, and can be synthesized according to methods known in the art (e.g., the method described in US2005-0238649A1). The binding unit may be a “cleavable binding unit” that facilitates the release of a drug within a cell. For example, acid-unstable binding units (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) binding units, photo-unstable binding units, dimethyl binding units, or disulfide-containing binding units can be used (Char I et al. Cancer Research 52:127-131, 1992; US Patent No. 5,208,020).

[0047] Depending on the mechanism of intracellular drug release, the "binding units" or "binding units of antibody-drug conjugates" described herein can be divided into two types: incleavable binding units and cleavable binding units. The drug release mechanism of antibody-drug conjugates containing incleavable binding units is as follows: After the conjugate binds to an antigen and is taken up by the cell, the antibody is enzymatically hydrolyzed within the lysosome, releasing an active molecule consisting of a small molecule drug, a binding unit, and antibody amino acid residues. This change in the drug molecule structure does not reduce its cytotoxicity, and because the active molecule is charged (amino acid residues), it does not penetrate neighboring cells. Therefore, such drugs do not kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13).

[0048] The term "ligand-drug conjugate" refers to an antibody binding to a biologically active drug via a stable binding unit. In the present invention, "ligand-drug conjugate" refers to an antibody-drug conjugate (ADC), preferably a monoclonal antibody or antibody fragment, which is bound to a biologically active toxic drug via a stable binding unit.

[0049] The three-letter and one-letter amino acid codes used herein are as described in J.boy.Chem.1968,243,3558.

[0050] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, and includes linear or branched groups having 1 to 20 carbon atoms, preferably alkyl groups having 1 to 12 carbon atoms, more preferably alkyl groups having 1 to 10 carbon atoms, and most preferably alkyl groups having 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, and 5-methylhexyl. Examples include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and their branched isomers.More preferably, the alkyl group is a lower alkyl group having 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl. The alkyl group may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available bond point. The substituents are preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxy groups.

[0051] The term "substituted alkyl" refers to an alkyl group in which hydrogen atoms are replaced by substituents. Unless otherwise specified in the context, substituents on an alkyl group are selected from -halogen, -OR', -NR'R'', -SR', -SIR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -CN and -NO2. The number of substituents is 0-(2m'+1), where m' is the total number of carbon atoms in the group. R', R'', and R''' are each independently hydrogen and unsubstituted carbon. 1-8An alkyl group, an unsubstituted aryl group, an aryl group substituted with 1 - 3 halogens, an unsubstituted C 1-8 alkyl group, C 1-8 an alkoxy group or C 1-8 a thioalkoxy group, or an unsubstituted aryl group - C 1-4 alkyl group. When R' and R'' are attached to the same nitrogen atom, they can together form a 3 -, 4 -, 5 -, 6 - or 7 - membered ring with this nitrogen atom. For example, -NR'R'' includes a 1 - pyrrolidinyl group and a 4 - morpholinyl group.

[0052] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O and S. The alkyl group is synonymous with the above.

[0053] The term "alkylene" refers to a saturated straight - chain or branched aliphatic hydrocarbon group having two residues obtained by removing two hydrogen atoms from the same or different carbon atoms of the parent alkane, including straight - chain or branched groups having 1 - 20 carbon atoms, preferably 1 - 12 carbon atoms, more preferably 1 - 6 carbon atoms alkylene groups. Non - limiting examples of alkylene groups include a methylene group (-CH2-), 1,1 - ethylene group (-CH(CH3)-), 1,2 - ethylene group (-CH2CH2-), 1,1 - propylene group (-CH(CH2CH3)-), 1,2 - propylene group (-CH2CH(CH3)-), 1,3 - propylene group (-CH2CH2CH2-), 1,4 - butylene group (-CH2CH2CH2CH2-) and 1,5 - butylene group (-CH2CH2CH2CH2CH2-), but are not limited thereto. The alkylene group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available bonding point. The substituents are preferably each independently selected from one or more of an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxyl group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, an oxy group.

[0054] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl) groups. Alkyl group or cycloalkyl group is synonymous with the above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy groups may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available bond point. The substituent is preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0055] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. A cycloalkyl ring contains 3-20, preferably 3-12, more preferably 3-10, and most preferably 3-8 carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl. Monocyclic cycloalkyl groups include spiro, condensed, or bridged cycloalkyl groups.

[0056] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3-20 ring-forming atoms, one or more of which are nitrogen, oxygen, and S(O). mThe heteroatom is selected from (where m is an integer between 0 and 2), but the -OO-, -OS-, or -SS- ring portion is excluded, and the remaining ring constituent atoms are carbon. Preferably, it contains 3 to 12 ring constituent atoms, of which 1 to 4 are heteroatoms. More preferably, the cycloalkyl ring contains 3 to 10 ring constituent atoms. Examples of monocyclic heterocyclyl groups include, but are not limited to, pyrrolidinyl alkyl groups, piperidinyl groups, piperazinyl groups, morpholinyl groups, thiomorpholinyl groups, and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro, condensed, or bridge ring heterocyclyl groups.

[0057] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group in which one atom (called a spiro atom) is shared between monorings ranging from 5 to 20 members, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the other ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of spiro atoms shared between the rings, spiroheterocyclyls are classified as monoheterocyclyls, dispiroheterocyclyls, or polyspiroheterocyclyls, preferably monospiroheterocyclyls or dispiroheterocyclyls, more preferably 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 5-member, or 5-member / 6-member monospiroheterocyclyls.

[0058] The term "cycloalkylalkyl" refers to a molecule in which one or more alkyl groups, preferably one cycloalkyl group, are substituted. Alkyl and cycloalkyl are synonymous with the terms used above.

[0059] The term "alkyl halide" refers to a molecule in which one or more halogens are substituted for an alkyl group. The term "alkyl group" is synonymous with the above.

[0060] The term "deuterated alkyl" refers to a molecule in which an alkyl group is substituted with one or more deuterium atoms. The term "alkyl group" is synonymous with the above.

[0061] The term "hydroxyl" refers to the -OH group.

[0062] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0063] The term "amino" refers to -NH2. The term "nitro" refers to -NO2.

[0064] The term "amide" refers to a -C(O)N-(alkyl) or (cycloalkyl) group, and alkyl and cycloalkyl are synonymous with the above.

[0065] The term "carboxylic acid ester" refers to a -C(O)O-(alkyl) or (cycloalkyl) group, and alkyl and cycloalkyl are synonymous with the above.

[0066] The present invention further includes deuterated forms. Each hydrogen atom bonded to a carbon atom can be independently substituted with a deuterium atom. Those skilled in the art can synthesize deuterated compounds according to relevant literature. When synthesizing deuterated compounds, commercially available deuterated starting materials may be used, or they may be synthesized using deuterated reagents by conventional methods. Deuterated reagents include, but are not limited to, deuterated methanol, deuterium hydroxide, deuterated borane, trideuterated boranetetrahydrofuran solution, lithium aluminum deuterated, deuterated iodoethane, and deuterated iodomethane.

[0067] The term "antibody" refers to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence in the constant region of the heavy chain of immunoglobulins differ, their antigenicity also differs. This allows immunoglobulins to be classified into five types or isotypes: IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are the μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be classified into different subclasses depending on the differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified into κ or λ chains depending on the differences in the constant region. Each of the five types of Ig can have either a κ or λ chain. The antibodies described in this invention are preferably specific antibodies against cell surface antigens on target cells.In non-limiting embodiments, the antibody is one or more of an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-CD70 antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-claudin 18.2 antibody, an anti-Mesothelin antibody, an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 (ErbB3) antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-NectIn 4 antibody, an anti-TROP-2 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FRα antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin αv antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody or an anti-CD123 antibody. Preferably, it is Trastuzumab (trade name HerceptIn), Pertuzumab (also called 2C4; trade name Perjeta), NImotuzumab (trade name: Taishengxing), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab.

[0068] The term "solvate" refers to a pharmaceutically acceptable solvate formed from the ligand-drug conjugate of the present invention and one or more solvent molecules. Examples of solvent molecules include, but are not limited to, water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0069] The term "drug load" refers to the average number of cytotoxic agents loaded onto each antibody in the Formula I molecule, and may be expressed as a ratio of drug amount to antibody amount. Within the drug load range, 0-12, preferably 1-10 cytotoxic agents (D) can be bound to each antibody (Ab). In embodiments of the present invention, the drug load is denoted by n, and may be an average value such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average number of drugs on each ADC molecule after the coupling reaction can be measured by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC.

[0070] In one embodiment of the present invention, a cytotoxic drug is bound to the ε-amino of the N-terminal amino and / or lysine residue of the ligand via a binding unit. Generally, the number of drug molecules that can bind to an antibody in a coupling reaction is smaller than the theoretical maximum.

[0071] The amount of ligand-cytotoxic drug conjugates can be controlled by the following non-restrictive methods. (1) Control the molar ratio of the binding unit reagent to the monoclonal antibody. (2) Control the reaction time and temperature. (3) Select different reaction reagents. For the manufacture of typical pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0072] The terms “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refer to a salt of the ligand-drug conjugate of the present invention or a salt of a compound described in the present invention. Such salts are safe and effective when used in mammals and possess appropriate biological activity. The ligand-drug conjugate of the present invention has at least one carboxyl and can therefore form salts with bases. Non-restrictive examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, and the like.

[0073] The terms “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refer to a salt of the ligand-drug conjugate of the present invention or a salt of a compound described in the present invention. Such salts are safe and effective when used in mammals and have appropriate biological activity. The antibody-drug conjugate compounds of the present invention contain at least one amino group and can therefore form salts with acids. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, hydrobroms, hydroiodides, sulfates, bisulfates, citrates, acetates, succinates, ascorbicates, oxalates, nitrates, sorbates, hydrogen phosphates, dihydrogen phosphates, salicylates, hydrogen citrates, tartrates, maleates, fumarates, formates, benzoates, mesylates, ethanesulfons, benzenesulfons, and p-toluenesulfons.

[0074] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. Acidic amino acid molecules typically have one or more acidic groups, such as carboxyl groups, and structurally, they can be effectively ionized to become negative ions, thereby improving their hydrophilicity. Acidic amino acids can be divided into natural and unnatural amino acids.

[0075] "Natural amino acids" refer to amino acids obtained through biosynthesis. Natural amino acids are generally L-type, but there are exceptions, such as glycine, which includes both naturally occurring forms and those synthesized within the body.

[0076] "Non-natural amino acids" refer to amino acids obtained through synthesis.

[0077] The present invention will be further described below with reference to specific examples, but please understand that these examples are used solely to illustrate the present invention and are not intended to limit the scope of the invention. In the following examples, test methods for which specific conditions are not indicated generally follow conventional conditions or conditions proposed by the manufacturer. Unless otherwise specified, all percentages, proportions, ratios or parts are by weight.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly known in the art. Furthermore, any methods and materials similar or equivalent to those described herein are applicable to the methods of the present invention. The preferred methods and materials described herein are illustrative only.

[0079] Example 1: Synthesis of Exatecan enantiomer compound 1 [ka] In a 1 L round-bottom flask, compound SM-1 (N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide; purchased) (6.25 g, 25.0 mmol), SM-2 ((S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran-O[3,4-F]indoxazine-3,6,10(4H)-ketone; purchased) (7.25 g, 27.5 mmol), and pyridinium p-toluenesulfonate (1.26 g, 5.0 mmol) were added. 500 mL of toluene was added, and the mixture was reacted under heating and reflux for 5 hours. The reaction was monitored by HPLC until the concentration of SM-2 fell below 10%, at which point the reaction was stopped. The solution was cooled, concentrated under reduced pressure until the volume was halved, crystallized in an ice bath with stirring for 2 hours, filtered, the filter cake was washed in three separate 30 mL toluene washes, and vacuum dried to obtain compound Ac-1 (10.68 g, 89%). The LC-MS result was [M+H] + The value was 478.2.

[0080] Ac-1 (10.68 g, 22.5 mmol), methanesulfonic acid (55 mL), water (110 mL), and toluene (55 mL) were mixed in a 1 L round-bottom flask and heated under reflux. After 4 hours, the reaction was monitored by TLC, and after the starting materials had completely reacted, the reaction system was cooled to room temperature. 500 mL of methanol was slowly added dropwise to the reaction system at room temperature, and stirring was continued. After the addition was complete, the mixture was stirred overnight, and the reaction system was filtered by suction. The filtrate cake was washed with methanol, the filtrate was collected, and the filtrate cake was dried to obtain exatecan, an isomer of compound 1, which was a grayish-white solid. The filtrate was concentrated under reduced pressure, purified by preparative liquid chromatography, and freeze-dried to obtain a light brown solid (5.07 g, 41%). The LC-MS result was [M+H] + The value was 436.2.

[0081] Example 2: Synthesis of Compound 2 [ka] Compound 1 (in the form of trifluoroacetate) (100.0 mg, 0.188 mmol), glycolic acid (43.0 mg, 0.565 mmol), HATU (107.2 mg, 0.282 mmol), HOBt (38.1 mg, 0.282 mmol), and 5 ml of ultra-dry DMF were placed in a 25 ml round-bottom flask, and the flask neck was sealed. The flask was placed in an ice bath and stirred for 10 minutes, after which DIEA (94 μL, 0.565 mmol) was added. The progress of the reaction was monitored by TLC, and the reaction was allowed to proceed for approximately 3 hours. After the reaction was completely finished, the reaction mixture was separated, the separate solution was concentrated under vacuum at 35°C, and freeze-dried to obtain a white solid compound 2 (63.8 mg, yield 69.7%). The LC-MS result was [M+H] + The value was 493.9. 11H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 9.0 Hz, 1H), 7.68 (d, J = 10.9 Hz, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 5.53 (dt, J = 9.1, 6.0 Hz, 1H), 5.41 (s, 2H), 5.12 (d, J = 19.1 Hz, 1H), 4.92 (d, J = 18.9 Hz, 1H), 3.99 (s, 2H), 3.10 (qt, J = 16.7, 6.1 Hz, 2H), 2.29 (d, J = 1.8 Hz, 3H), 2.16 (q, J = 6.4 Hz, 2H), 2.00 - 1.81 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H).

[0082] Example 3: Synthesis of Compound 3

Chemical Structure

[0083] 1H NMR(400 MHz, DMSO-d6) δ 8.46(d, J=9.2 Hz, 1H), 7.71(d, J=10.9 Hz, 1H), 7.29(s, 1H), 6.53(s, 1H), 5.68(d, J=4.9 Hz, 1H), 5.54(q, J=7.3 Hz, 1H), 5.42(s, 2H), 5.20(d, J=19.0 Hz, 1H), 4.93(d, J=18.9 Hz, 1H), 4.20-4.08(m, 1H), 3.25-3.01(m, 2H), 2.32(s, 3H), 2.14(q, J=6.5 Hz, 2H), 1.96-1.80(m, J=7.1 Hz, 2H), 1.42(d, J=6.8 Hz, 3H), 0.90(t, J=7.2 Hz, 3H).

[0084] Example 4: Synthesis of Compound 4 [ka] Compound 1 (in the form of trifluoroacetate) (100.0 mg, 0.188 mmol), D-lactic acid (56.6 mg, 0.565 mmol), HATU (107.2 mg, 0.282 mmol), HOBt (38.1 mg, 0.282 mmol), and 5 ml of ultra-dry DMF were added to a 25 ml round-bottom flask, and the flask neck was sealed. The flask was placed in an ice bath and stirred for 10 minutes, after which DIEA (94 μL, 0.565 mmol) was added. The reaction was monitored by TLC and allowed to proceed for approximately 3 hours. After the reaction was completely finished, saturated ammonium chloride aqueous solution was added to the reaction mixture, and a pale yellow crude product was obtained by suction filtration. The crude product was scraped off and separated using a TLC preparative plate, and the solid was slurried with dichloromethane and methanol to purify it and obtain 16.8 mg of pale yellow solid compound 4 (yield 18.4%). The LC-MS result was [M+H] + The value was 508.2.

[0085] Example 5: Synthesis of Compound 5 and Compound 6 [ka] Compound 1 (in the form of trifluoroacetate) (200 mg, 0.377 mmol), trifluorolactic acid (162.8 mg, 1.13 mmol), HATU (214.8 mg, 0.565 mmol), HOBt (76.3 mg, 0.565 mmol), and 5 ml of ultra-dry DMF were placed in a 25 ml round-bottom flask, and the flask neck was sealed. The flask was placed in an ice bath and stirred for 10 minutes, after which DIEA (187 μL, 1.13 mmol) was added. The progress of the reaction was monitored by TLC, and the reaction was allowed to proceed for approximately 6 hours. After the reaction was completely finished, the reaction mixture was separated and purified by preparative liquid chromatography, the preparatives were concentrated under vacuum at 35°C, and freeze-dried to obtain 49.7 mg of pale yellow solid compound 5 (yield 48.7%) and 60.5 mg of pale yellow solid compound 6 (yield 59.3%).

[0086] Compound 5: The LC-MS results are [M+H] + The value was 562.2. 1 H NMR(400 MHz, DMSO-d6) δ 9.00(d, J=8.7 Hz, 1H), 7.78(d, J=10.9 Hz, 1H), 7.31(s, 1H), 7.19(d, J=6.9 Hz, 1H), 6.54(s, 1H), 5.59(dt, J=10.1, 5.3 Hz, 1H), 5.43(s, 2H), 5.16(q, J=19.2 Hz, 2H), 4.63(p, J=7.6 Hz, 1H), 3.14(s, 2H), 2.38(s, 3H), 2.16(p, J=7.1 Hz, 2H), 1.87(hept, J=7.1 Hz, 2H), 0.88(t, J=7.3 Hz, 3H).

[0087] Compound 6: The LC-MS results are [M+H] + The value was 562.2. 1H NMR(400 MHz, DMSO-d6) δ 8.97(d, J=8.6 Hz, 1H), 7.78(d, J=11.0 Hz, 1H), 7.31(s, 1H), 7.26(d, J=6.7 Hz, 1H), 6.54(s, 1H), 5.57(dt, J=9.5, 5.3 Hz, 1H), 5.43(s, 2H), 5.24-5.03(m, 2H), 4.63(p, J=7.4 Hz, 1H), 3.14(t, J=6.5 Hz, 2H), 2.38(d, J=1.9 Hz, 3H), 2.28-2.07(m, 2H), 1.95-1.78(m, J=7.1 Hz, 2H), 0.88(t, J=7.3 Hz, 3H).

[0088] Example 6: Synthesis of Compound 7 and Compound 8 [ka] Compound 1 (in the form of trifluoroacetate) (200 mg, 0.377 mmol), 1-hydroxycyclopropanecarboxylic acid (131.2 mg, 1.13 mmol), HATU (214.8 mg, 0.565 mmol), HOBt (76.3 mg, 0.565 mmol), and 5 ml of ultra-dry DMF were added to a 25 ml round-bottom flask, and the flask neck was sealed. The flask was placed in an ice bath and stirred for 10 minutes, after which DIEA (187 μL, 1.13 mmol) was added. The progress of the reaction was monitored by TLC, and the reaction was allowed to proceed for approximately 6 hours. After the reaction was completely finished, water was added to the reaction solution to precipitate the solid, which was washed twice with saturated aqueous ammonium chloride solution, once with pure water, dried by suction, and then dissolved with dichloromethane and methanol. The compounds were scraped and separated using a TLC preparative plate to obtain 81.0 mg of pale yellow solid compound 7 (yield 82.4%) and 60.0 mg of pale yellow solid compound 8 (yield 61.8%).

[0089] Compound 7: LC-MS results are [M+H] + The value was 534.2. 1H NMR(400 MHz, d6DMSO) δ 8.40(d, J=9.0 Hz, 1H), 7.78-7.62(m, 1H), 7.29(s, 1H), 6.52(s, 1H), 5.58(dt, J=5.3, 1.4 Hz, 1H), 5.53(q, J=7.5 Hz, 1H), 5.42(s, 2H), 5.26-4.91(m, 2H), 3.62(t, J=5.8 Hz, 1H), 3.23-3.02(m, 2H), 2.30(d, J=3.2 Hz, 3H), 2.14(q, J=7.9, 7.0 Hz, 2H), 1.90(p, J=7.0 Hz, 2H), 1.29-1.19(m, 3H), 0.91(t, J=7.3 Hz, 3H), 0.57-0.37(m, 4H). Compound 8: LC-MS results are [M+H] + The value was 534.2.

[0090] Example 7: Synthesis of Compound 9 [ka]

[0091] Compound 1 (in the form of trifluoroacetate) (100.0 mg, 0.188 mmol), 1-(hydroxymethyl)cyclobutanecarboxylic acid (43.0 mg, 0.565 mmol), HATU (107.2 mg, 0.282 mmol), HOBt (38.1 mg, 0.282 mmol), and 5 ml of ultra-dry DMF were placed in a 25 ml round-bottom flask, and the flask neck was sealed. The flask was placed in an ice bath and stirred for 10 minutes, after which DIEA (94 μL, 0.565 mmol) was added. The progress of the reaction was monitored by TLC, and the reaction was allowed to proceed for approximately 6 hours. After the reaction was completely finished, saturated ammonium chloride aqueous solution was added to the reaction mixture to precipitate the solid, which was dried by suction to obtain the solid. The solid was washed twice with water, once with ethyl acetate, and once with dichloromethane. 100.0 mg of grayish-white solid compound 9 (yield 98.0%) was obtained. The LC-MS result was [M+H] + The value was 548.2. 1H NMR(400 MHz, DMSO-d6) δ 7.99(d, J=8.6 Hz, 1H), 7.69(d, J=10.9 Hz, 1H), 7.29(s, 1H), 6.52(s, 1H), 5.53(dt, J=8.7, 5.5 Hz, 1H), 5.42(s, 2H), 5.21-4.93(m, 3H), 3.63(d, J=4.8 Hz, 2H), 3.10(t, J=6.2 Hz, 2H), 2.39-2.24(m, 5H), 2.12(q, J=6.3 Hz, 2H), 1.99-1.70(m, 6H), 0.91(t, J=7.3 Hz, 3H).

[0092] Example 8: Synthesis of Compound M1 [ka] In a 5000 mL single-neck flask, N-fluorenylmethyloxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq; purchased), lead tetraacetate (175 g, 553 mmol, 1.4 eq), 2000 mL dry tetrahydrofuran, and 670 mL toluene were added and mixed thoroughly. The mixture was heated to 85 °C under nitrogen gas protection and reacted for 2.5 hours. After monitoring by TLC and confirming that the starting materials had reacted completely, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound M1 (91 g). LC-MS: [M + NH4] + 386.0.

[0093] Example 9: Synthesis of Compound M2 [ka] Compound SM-3 (49.9g, 100.0 mmol, 1.0 eq, synthesized by the method disclosed in our patent application CN108452321), pentafluorophenol (18.5g, 110.0 mmol, 1.1 eq), DCC (20.64g, 110.0 mmol, 1.1 eq), and THF (500mL) were added to a 1000mL single-neck flask. The mixture was allowed to react at room temperature for 1 hour (monitoring for complete reaction by TLC), insoluble matter was removed by filtration, and the resulting filtrate was recorded as filtrate A. It was stored at 2-8°C and prepared for use (the solvent was taken directly and used. It was calculated and used as 0.2M). LC-MS: [M+H] + 565.2.

[0094] Example 10: Synthesis of compound Ln-D1 [ka]

[0095] Step 1: Synthesis of Compound 1a In a 500 ml round-bottom flask, M1 (15.00 g, 40.75 mmol) and 150 ml of THF were placed and stirred in an ice bath for 15 minutes. p-toluenesulfonic acid monohydrate (775.0 mg, 4.08 mmol) was added and stirred for another 10 minutes. A constant-pressure dropping funnel was attached to the top of the flask and benzyl glycolate (11.6 ml, 81.50 mmol) was added dropwise. After the addition was complete, the mixture was stirred in an ice bath and allowed to react completely for approximately 3 hours. After confirming that all the starting materials had been used by TLC, the reaction was quenched with sodium bicarbonate, the mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography. Separation yielded 9.06 g of white solid (yield 46.9%).

[0096] Step 2: Synthesis of Compound 1b 1a (5.34 g, 11.26 mmol) and 25 ml of DMF were placed in a 50 ml round-bottom flask (No. 1) and stirred in an ice bath for 15 minutes. Triethylamine (1854 uL, 12.39 mmol) was added dropwise, and the reaction was allowed to proceed for approximately 5 hours. After monitoring for the complete removal of the starting materials by TLC, the mixture was prepared for use once the reaction was complete. 20 ml of DMF was placed in a 100 ml round-bottom flask (No. 2) and stirred in an ice bath for 15 minutes. The reaction solution from flask No. 1, which had completely reacted, was added dropwise to reaction flask No. 2. The complete removal of the starting materials was monitored by HPLC, and after the reaction was completely finished, the mixture was separated by HPLC preparative sampling. The mixture was freeze-dried to obtain the product: 4.83 g of white solid (yield 66.3%).

[0097] Step 3: Synthesis of Compound 1c 1b (500.0 mg, 0.772 mmol), 5% Pd / C (500.0 mg, 100% m), and 10 ml of DCM were placed in a 50 mL single-neck flask. A hydrogen balloon was added to purge the solution with hydrogen, and the mixture was reacted at room temperature for approximately 3 hours. After confirming complete reaction by HPLC, the solution was filtered to obtain the filtrate. Crude product 1c was obtained and used directly in the next reaction.

[0098] Step 4: Synthesis of compound 1d Crude product 1c was placed in an ice bath, DIPEA (0.14 mL, 0.82 mmol) was added, followed by compound M2 (4.0 mL, 0.8 mmol). After addition, the mixture was heated to room temperature and reacted for 1 hour. The reaction was monitored by HPLC, the liquid phase was purified, and the preparative solution was obtained. This solution was then freeze-dried to obtain 370.2 mg of white solid (yield 59.7%). LC-MS: [M+H] + 804.4.

[0099] Step 5: Synthesis of Compound 1e In a 10 ml single-neck flask, 1d (260.0 mg, 0.281 mmol), compound 1 (149.3 mg, 0.281 mmol), HATU (160.4 mg, 0.422 mmol), HOBt (57.0 mg, 0.422 mmol), and 5 ml of DMF were placed. The mixture was stirred in an ice bath for 10 minutes, and after confirming complete reaction by HPLC, the mixture was separated and the fraction was freeze-dried to obtain 368.0 mg of yellow solid (yield 97.3%). LC-MS: [M+H] + 1221.5

[0100] Step 6: Synthesis of compound Ln-D1 1e (368 mg, 0.289 mmol), zinc bromide (1301.0 mg, 5.78 mmol), and 15 ml of nitromethane were added to a 25 ml single-neck flask and stirred at room temperature. After confirming that the starting materials had reacted completely by HPLC, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 120 mg of yellow solid (yield 38.5%). LC-MS: [M+H] + 1065.4.

[0101] Example 11: Synthesis of compound Ln-D2 [ka]

[0102] Step 1: Synthesis of Compound 2a Add M1 (7.45 g, 20.3 mmol) and 120 ml of THF to a 250 ml round-bottom flask and stir in an ice bath for 15 minutes. Add p-toluenesulfonic acid monohydrate (385.0 mg, 2.03 mmol) and continue stirring for 10 minutes. Attach a constant-pressure dropping funnel to the top of the flask and add benzyl glycolate (6.0 ml, 41.5 mmol) dropwise, controlling the rate to one drop every 2 seconds. After the addition is complete, continue stirring in an ice bath and allow the reaction to proceed completely for approximately 3 hours. After confirming that all starting materials have been used by TLC, the reaction is quenched with sodium bicarbonate, the mixture is extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain a 4.82 white solid (yield 49%).

[0103] Step 2: Synthesis of Compound 2b 2a (2.53 g, 5.20 mmol) and 10 ml of DMF were placed in a 25 ml round-bottom flask (No. 1) and stirred in an ice bath for 15 minutes. DBU (900 uL, 6.0 mmol) was added dropwise and the reaction was allowed to proceed for approximately 0.5 hours. The reaction was monitored by TLC to ensure that all starting materials were consumed, and the mixture was prepared for use after complete reaction. M4 (2.31 g, 5.6 mmol), PyBop (3.50 g, 6.8 mmol), DIEA (1115 uL, 6.8 mmol) and 10 ml of DMF were placed in a 50 ml round-bottom flask (No. 2) and stirred in an ice bath for 15 minutes. The reaction solution from flask No. 1, which had completely reacted, was added dropwise to reaction flask No. 2. The reaction was monitored by HPLC to ensure that all starting materials were consumed, and after the reaction was completely finished, the mixture was separated by HPLC preparative sampling. Freeze-drying yielded 2.53 g of white solid (yield 74%).

[0104] Step 3: Synthesis of Compound 2c 2b (500.0 mg, 0.76 mmol), 5% Pd / C (500.0 mg, 100% m) and 10 ml of DCM were placed in a 50 mL single-neck flask, hydrogen purging was performed by adding a hydrogen balloon, and the mixture was reacted at room temperature for approximately 3 hours. After confirming complete reaction by HPLC, the mixture was filtered to obtain the filtrate. Crude product 2c was used directly in the next reaction.

[0105] Step 4: Synthesis of compound 2d Crude product 2c was placed in an ice bath, DIPEA (0.12 mL, 0.70 mmol) was added, followed by compound M2 (4.0 mL, 0.8 mmol). After addition, the mixture was heated to room temperature and reacted for 1 hour. The reaction was monitored by HPLC, the liquid phase was purified, and the preparative solution was obtained and freeze-dried to yield 378.2 mg of white solid (yield 62%). LC-MS: [M+H] + 818.3.

[0106] Step 5: Synthesis of Compound 2e In a 10 ml single-neck flask, combine 2d (228 mg, 0.28 mmol), compound 1 (148.3 mg, 0.28 mmol), HATU (160.1 mg, 0.42 mmol), HOBt (57.2 mg, 0.42 mmol), and 5 ml of DMF. Stir in an ice bath for 10 minutes, then add DIEA (140 uL, 0.84 mmol) dropwise and continue the reaction. After confirming complete reaction by HPLC, the mixture is separated and freeze-dried to obtain 269.5 mg of yellow solid (78% yield). LC-MS: [M+H] + 1235.5.

[0107] Step 6: Synthesis of compound Ln-D2 2e (269.5 mg, 0.22 mmol), zinc bromide (1012.9 mg, 0.44 mmol), and 10 ml of nitromethane were placed in a 25 ml single-neck flask and stirred at room temperature. After confirming that the starting materials had reacted completely by HPLC, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 96.3 mg of yellow solid (yield 41%). LC-MS: [M+H] + 1079.4.

[0108] Example 12: Synthesis of compound Ln-D3 [ka] Compound L-D3 (93.8 mg) was obtained by the synthesis route and method of Example 11. LC-MS: [M+H] + 1079.4.

[0109] Example 13: Synthesis of compounds Ln-D4 and Ln-D5 [ka]

[0110] Step 1: Synthesis of compound 3a In a 100 ml round-bottom flask, M1 (2.87 g, 7.79 mmol), benzyl trifluorolactic acid (3.65 g, 15.59 mmol), anhydrous zinc acetate (2.86 g, 15.59 mmol), and 30 ml of toluene were added and the mixture was reacted at 100°C under nitrogen gas protection. The reaction was monitored by TLC and allowed to proceed for approximately 5 hours. After complete reaction, the mixture was filtered, and the filtrate was concentrated to obtain the crude oily product. The crude product was purified by silica gel column chromatography to obtain 1.51 g of a white solid (yield 35.7%).

[0111] Step 2: Synthesis of Compound 3b 3a (2.82g, 5.20 mmol) and 10 ml of DMF were placed in a 50 ml round-bottom flask (size 1) and stirred in an ice bath for 15 minutes. DBU (0.949g, 6.24 mmol) was added dropwise, and the reaction was allowed to proceed for approximately 0.5 hours. The reaction was monitored by TLC to ensure that all starting materials were used, and the mixture was prepared for use after complete reaction. In a 50 ml round-bottom flask (No. 2), M4 ((2.36 g, 5.71 mmol), PyBop (3.25 g, 6.24 mmol), HOBt (0.84 g, 6.24 mmol), DIEA (1000 uL, 6.24 mmol) and 10 ml of DMF were placed and stirred in an ice bath for 15 minutes. The reaction solution in flask No. 1, which had completely reacted, was added dropwise to reaction flask No. 2. The reaction was monitored by HPLC to ensure that all starting materials were used, and after the reaction was completely finished, the mixture was separated by HPLC preparative extraction. The preparative solution was extracted three times by DCM, dried over anhydrous sodium sulfate, the organic phase was concentrated, and vacuum drying was performed to obtain 2.32 g of white solid (yield 62%).

[0112] Step 3: Synthesis of Compound 3c 3b (500.0 mg, 0.70 mmol), 5% Pd / C (500.0 mg, 100% m), and 10 ml of DMF were placed in a 50 mL one-neck flask and reacted at room temperature for approximately 3 hours. After confirming complete reaction by HPLC, the mixture was filtered to obtain the filtrate, and the crude product 3c was used directly in the next reaction.

[0113] Step 4: Synthesis of compound 3D Crude product 3c was placed in an ice bath, DIPEA (0.12 mL, 0.70 mmol) was added, followed by the addition of compound M2 (4.0 mL, 0.8 mmol). After addition, the temperature was raised to room temperature and the reaction was allowed to proceed for 1 hour. The reaction was monitored by HPLC, the liquid phase was purified, and the preparative solution was obtained and freeze-dried to yield 280.0 mg of white solid (yield 46%). LC-MS: [M+H] + 872.3.

[0114] Step 5: Synthesis of Compound 3e In a 10 ml single-neck flask, combine 3d (280.0 mg, 0.241 mmol), compound 1 (142.2 mg, 0.241 mmol), HATU (137.6 mg, 0.362 mmol), HOBt (38.9 mg, 0.362 mmol), and 5 ml of DMF. Stir in an ice bath for 10 minutes, then add DIEA (120 uL, 0.723 mmol) dropwise and continue the reaction. After confirming complete reaction by HPLC, separate the solution, freeze-dry to obtain 154.1 mg of yellow solid 3e (yield 73.6%; LC-MS: [M+H]). + 1289.5), 62.5 mg yellow solid iso-3e (29.8% yield, LC-MS: [M+H] + 1289.5) was obtained.

[0115] Step 6: Synthesis of compounds Ln-D4 and Ln-D5 3e (154.1 mg, 0.118 mmol), zinc bromide (532.5 mg, 2.36 mmol), and 10 ml of nitromethane were placed in a 25 ml single-neck flask and stirred at room temperature. After confirming that the starting materials had reacted completely by HPLC, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 82.0 mg of yellow solid (yield 60%). LC-MS: [M+H] + 1133.4.

[0116] In a 25 ml single-neck flask, iso-3e (62.5 mg, 0.048 mmol), zinc bromide (216.0 mg, 0.959 mmol), and 7 ml of nitromethane were placed and stirred at room temperature. After confirming that the starting materials had reacted completely by HPLC, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 96.3 mg of yellow solid (yield 41%). LC-MS: [M+H] + 1133.4.

[0117] Example 14: Synthesis of compounds Ln-D6 and Ln-D7 [ka]

[0118] Step 1: Synthesis of Compound 4a In a 250 ml round-bottom flask, M1 (3.75 g, 10.19 mmol) and 50 ml of THF were placed and stirred in an ice bath for 15 minutes. p-toluenesulfonic acid monohydrate (194.0 mg, 1.02 mmol) was added and the mixture was stirred for another 10 minutes. A constant-pressure dropping funnel was attached to the top of the flask, and 2-cyclopropyl-2-hydroxyacetate benzyl (4.34 g, 20.4 mmol) was added dropwise and allowed to react completely for approximately 6 hours. After confirming that all starting materials had been used by TLC, the reaction was quenched with sodium bicarbonate, the mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain 2.13 g of white solid (yield 41%).

[0119] Step 2: Synthesis of Compound 4b 4a (2.80g, 5.38 mmol) and 10 ml of DMF were placed in a 50 ml round-bottom flask (No. 1) and stirred in an ice bath for 15 minutes. DIEA (0.981g, 6.45 mmol) was added dropwise, and the reaction was allowed to proceed for approximately 0.5 hours. The reaction was monitored by TLC to ensure all starting materials were consumed, and the mixture was prepared for use after complete reaction. M4 (2.22g, 5.38 mmol), PyBop (3.35g, 6.45 mmol), HOBt (0.87g, 6.45 mmol), DIEA (1000uL, 6.45 mmol) and 10 ml of DMF were placed in a 100 ml round-bottom flask (No. 2) and stirred in an ice bath for 15 minutes. The reaction solution from flask No. 1, which had completely reacted, was added dropwise to reaction flask No. 2. The reaction was monitored by HPLC to ensure all starting materials were consumed, and after the reaction was completely finished, the mixture was separated by HPLC preparative sampling. Freeze-drying yielded 2.63 g of white solid (61% yield).

[0120] Step 3: Synthesis of Compound 4c 4b (1003.1 mg, 1.46 mmol), 5% Pd / C (1004.2 mg, 100% m), and 15 ml of DMF were placed in a 50 mL single-neck flask. A hydrogen balloon was added to purge the solution with hydrogen, and the mixture was reacted at room temperature for approximately 3 hours. After confirming complete reaction by HPLC, the mixture was filtered to obtain the filtrate, and the crude product 4c was used directly in the next reaction.

[0121] Step 4: Synthesis of compound 4d Crude product 4c was placed in an ice bath, DIPEA (0.26 mL, 1.6 mmol) was added, followed by compound M2 (8.0 mL, 1.6 mmol). The mixture was then heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, the liquid phase was purified, and the preparative solution was obtained. It was then freeze-dried to yield 945.1 mg of white solid (76% yield). LC-MS: [M+H] + 844.4.

[0122] Step 5: Synthesis of Compound 4e In a 10 ml single-neck flask, combine 4d (422.0 mg, 0.50 mmol), compound 1 (265.5 mg, 0.50 mmol), HATU (285.1 mg, 0.75 mmol), HOBt (101.3 mg, 0.75 mmol), and 5 ml of DMF. Stir in an ice bath for 10 minutes, then add DIEA (248 μL, 1.50 mmol) dropwise and continue the reaction. After confirming complete reaction by HPLC, the mixture was separated and freeze-dried to obtain 268.0 mg of yellow solid (42% yield). LC-MS: [M+H] + 1261.5.

[0123] Step 6: Synthesis of compounds Ln-D6 and Ln-D7 4d((268.0 mg, 0.204 mmol), zinc bromide (1168.1 mg, 5.187 mmol)) and 10 ml of nitromethane were placed in a 50 ml one-neck flask and stirred at room temperature. The reaction was monitored by HPLC, and after complete reaction, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 68.0 mg of Ln-D6 yellow solid (yield 28%, LC-MS: [M+H]). + 1105.4), Ln-D7 yellow solid 82.0 mg (yield 35%, LC-MS: [M+H] + 1105.4) was obtained.

[0124] Example 15: Synthesis of compound Ln-D8 [ka]

[0125] Step 1: Synthesis of compound 5a In a 250 ml round-bottom flask, M1 (3.74 g, 10.16 mmol) and 50 ml of THF were placed and stirred in an ice bath for 15 minutes. p-toluenesulfonic acid monohydrate (193.1 mg, 1.02 mmol) was added and the mixture was stirred for another 10 minutes. A constant-pressure dropping funnel was attached to the top of the flask, and 1-(hydroxymethyl)cyclobutanecarboxylate benzyl (4.59 g, 20.3 mmol) was added dropwise. After the addition was complete, the mixture was stirred in an ice bath and reacted completely in about 5 hours. After confirming that all the starting materials had been used by TLC, the reaction was quenched with sodium bicarbonate, the mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain 4.60 g of white solid (yield 82%).

[0126] Step 2: Synthesis of Compound 5b 5a (4.38 g, 8.29 mmol) and 20 ml of DMF were placed in a 50 ml round-bottom flask (No. 1) and stirred in an ice bath for 15 minutes. DBU (1363 uL, 9.11 mmol) was added dropwise and the reaction was allowed to proceed for approximately 0.5 hours. The reaction was monitored by TLC to ensure that all starting materials were consumed, and the mixture was prepared for use after complete reaction. M4 (3.42 g, 8.29 mmol), PyBop (5.17 g, 9.95 mmol), HOBt (1.34 g, 9.95 mmol), DIEA (1640 uL, 9.95 mmol) and 30 ml of DMF were placed in a 50 ml round-bottom flask (No. 2) and stirred in an ice bath for 15 minutes. The reaction solution from flask No. 1, which had completely reacted, was added dropwise to reaction flask No. 2. The reaction was monitored by HPLC to ensure that all starting materials were consumed, and after the reaction was completely finished, the mixture was separated by HPLC preparative sampling. Freeze-drying yielded 4.60 g of white solid (68% yield).

[0127] Step 3: Synthesis of Compound 5c 5b (1041.5 mg, 1.26 mmol), 5% Pd / C (1056.7 mg, 100% m) and 10 ml of DMF were placed in a 50 mL single-neck flask, hydrogen purging was performed by adding a hydrogen balloon, and the mixture was reacted at room temperature for approximately 3 hours. The reaction was monitored by HPLC, and after the reaction was completely finished, hydrogen gas was removed by sonication. The mixture was filtered to obtain the filtrate, and the crude product 5c was used directly in the next reaction.

[0128] Step 4: Synthesis of compound 5d Crude product 5c was placed in an ice bath, DIPEA (0.24 mL, 1.4 mmol) was added, followed by the addition of compound M2 (7.0 mL, 1.4 mmol). After addition, the mixture was heated to room temperature and reacted for 1 hour. The reaction was monitored by HPLC, the liquid phase was purified, and the preparative solution was obtained and freeze-dried to yield 803.5 mg of white solid (yield 64%). LC-MS: [M+H] + 858.4.

[0129] Step 5: Synthesis of Compound 5e In a 10 ml single-neck flask, 5d (201.3 mg, 0.203 mmol), M2 amino isomer (100.0 mg, 0.203 mmol), HATU (107.4 mg, 0.304 mmol), HOBt (38.2 mg, 0.304 mmol), and 5 ml of DMF were placed. The mixture was stirred in an ice bath for 10 minutes, and DIEA (248 μL, 1.50 mmol) was added dropwise, continuing the reaction. After confirming complete reaction by HPLC, the mixture was separated, and the separated solution was freeze-dried to obtain 219.1 mg of yellow solid (yield 84%). LC-MS: [M+H] + 1275.5.

[0130] Step 6: Synthesis of compound Ln-D8 5d (219.1 mg, 0.170 mmol), zinc bromide (765.3 mg, 3.40 mmol), and 20 ml of nitromethane were placed in a 50 ml single-neck flask and stirred at room temperature. The reaction was monitored by HPLC, and after complete reaction, the reaction mixture was concentrated and purified by HPLC preparative separation. The preparative solution was freeze-dried to obtain 120.2 mg of yellow solid (62% yield). LC-MS: [M+H] + 1119.4.

[0131] Example 16: Synthesis of compound Ln-D9 [ka]

[0132] Step 1: Synthesis of compound 1f 1b (500.0 mg, 0.772 mmol), 5% Pd / C (500.0 mg, 100% m), and 10 ml of DMF were placed in a 50 mL single-neck flask. Hydrogen purging was performed by adding a hydrogen balloon, and the mixture was reacted at room temperature for approximately 3 hours. The reaction was monitored by HPLC, and after the reaction was completely finished, hydrogen gas was removed by sonication. The mixture was filtered to obtain the filtrate, which yielded the crude product 1c. MC (280.1 mg, 0.9 mmol) and DIEA (235 mg, 1.8 mmol) were added sequentially to the filtrate under an ice bath. After adding the compounds under nitrogen gas protection, the mixture was heated to room temperature and reacted for 1 hour. The reaction was monitored by HPLC, purified by preparative liquid chromatography, and freeze-dried to obtain compound 1f (268.9 mg, 86%). MS:[M+H] + 617.2.

[0133] Step 2: Synthesis of compound Ln-D9 In a 10 ml single-neck flask, 1f (268.9 mg, 0.44 mmol), exatecan mesylate (233.8 mg, 0.44 mmol, purchased), HATU (190.5 mg, 0.50 mmol), HOBt (67.8 mg, 0.50 mmol), and 5 ml of DMF were placed. The mixture was stirred in an ice bath for 10 minutes, and DIEA (140 uL, 0.84 mmol) was added dropwise, continuing the reaction. After confirming complete reaction by HPLC, the mixture was separated, and the separated solution was freeze-dried to obtain 254.3 mg of yellow solid (yield 56%). LC-MS: [M+H] + 1034.4.

[0134] Example 17: Synthesis of the control compound The following compounds were synthesized by the methods described in patents CN111689980 and WO2020063676. [Table 1] JPEG0007860665000046.jpg218152

[0135] Example 18: General manufacturing method for ADC drugs by coupling. Antibody Ab with a monomer content exceeding 95% after pre-purification was transferred to phosphate buffer (10 mg / mL concentration) using an ultrafiltration centrifuge tube. 20 times the number of moles of antibody in TCEP was added, and the mixture was reacted at room temperature for 10 hours to break the disulfide bonds between antibody chains. 20 times the number of moles of antibody in binding unit toxin was added, and the mixture was reacted at room temperature for 2 hours. After the reaction was complete, the solution was replaced with PBS using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa to remove the unbound payload. The ADC sample after the solution change was filtered through a 0.22 micron sterile filter and stored until use.

[0136] The coupling payload compounds Ln-D1, Ln-D2, Ln-D4, Ln-D5, Ln-D6, Ln-D7, Ln-D8, and Ln-D9 were coupled with antibody molecules Ab (wherein antibody molecules Ab may be A: Trastuzumab antibody and B: Cetuximab antibody, respectively) using the general coupling method described in Example 18. The mean drug / antibody ratio (DAR) of the coupling product was measured by reverse-phase high-performance liquid chromatography. The table below shows the relevant information of the obtained ADC drug molecules and their correspondence with the payload molecules. [Table 2] JPEG0007860665000048.jpg193152

[0137] Example 19: LogP value test of camptothesin chiral derivatives The logP value is closely related to a compound's water solubility, membrane permeability, the ADME process in the body, and its affinity for its receptor, and plays an important role in the process by which compounds permeate biological membranes. The logP values ​​were obtained by measuring the distribution coefficients of camptothecin drugs in n-octane (oil) and water. The results of the drug studies are shown in the table below. [Table 3] JPEG0007860665000050.jpg216149JPEG0007860665000051.jpg224149JPEG0007860665000052.jpg47149

[0138] As can be seen from the above LogP results, the compounds of the present invention have lower LogP values ​​and better water solubility.

[0139] Example 20: Plasma agglutination and degradation test of ADC A sterile, specific volume of ADC sample was taken and added to human plasma from which human IgG had been removed so that the final concentration of ADC in the plasma was 0.6 mg / ml. Each ADC was prepared in three tubes and incubated in a 37°C water bath for 0 hours, 72 hours, and 7 days. After incubation, the ADC sample was removed, 100 μL of Protein A (MabSelect SuRe TMLX Lot:#10221479GE, washed with PBS) was added to each tube, and the mixture was agitated in a vertical mixer for 2 hours to allow adsorption. Washing, elution, and Tris-HCl neutralization steps were then performed, and the ADC obtained after incubation was subjected to SEC detection of the ADC samples incubated for specific times to measure the agglutination and degradation status. [Table 4] JPEG0007860665000054.jpg194157

[0140] The payload of ADC-1 is consistent with that of the commercially available antitumor drug Enhertu. As evidenced by the aggregation and degradation of ADC drugs in plasma, the aggregation and degradation of camptothesin chiral derivative ADCs are significantly lower than that of ADC-1, and they possess superior physical and chemical properties and can exist more stably in plasma.

[0141] Example 21: Cell activity test of camptothesin chiral derivatives The cytotoxic activity of camptothesin chiral derivatives was measured using the following experimental procedure. Chiral derivatized camptothecin drugs were added to A431, MDA-MB-468, SK-BR-3, Bxpc-3, and SW620 tumor cells, respectively, and cell viability was measured after 72 hours. Based on in vitro experiments with cells, cell viability, cytotoxicity, and programmed cell death induced by the camptothecin drugs of the present invention were measured.

[0142] The in vitro efficacy of camptothecin was measured using cell proliferation tests. CellTiter 96 (登録商標) The Aqueous One Solution Cell Proliferation Assay is a commercially available product (Promega Corp., Madison, WI). CellTiter96 (登録商標) Aqueous One Solution Cell Proliferation Assay(a) is a detection reagent that detects the number of viable cells in cell proliferation and cytotoxicity tests by colorimetric method. This reagent contains a novel tetrazole compound [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, internal salt; MTS] and an electron coupling agent (phenazine ethosulfate; PES). PES has enhanced chemical stability, allowing it to mix with MTS to form a stable solution. The MTS (Owen's reagent) in the reagent is bioreduced by cells to a colored formazan product that can then be dissolved in the culture medium. Such a conversion may be completed under the action of NADPH or NADH by dehydrogenases in metabolically active cells. When detecting, an appropriate amount of CellTiter96 (登録商標) Simply add the Aqueous One Solution Reagent directly to the culture plate wells, incubate for 1-4 hours, and read the absorbance value at 490 nm using a microplate reader.

[0143] The amount of formazan product detected at 490 nm is proportional to the number of viable cells in culture. Since the formazan product of MTS is soluble in tissue medium, CellTiter 96(登録商標) The Aqueous One Solution Assay has fewer operational steps compared to the MTT or INT methods.

[0144] In this invention, A431, MDA-MB-468, SK-BR-3, Bxpc-3, and SW620 are used as research systems for in vitro efficacy detection. Cells were uniformly inoculated at an appropriate cell density in a 96-well plate and incubated in a CO2 incubator (37°C). After 24 hours, the cell condition was confirmed to be normal under a microscope, and the camptothecin drug was added. The camptothecin drug (initial concentration 1 μM, 7-fold dilution, 8 concentration points; the last two columns were for the control group (i.e., cells + medium, no drug treatment) and the blank group (i.e., no cells, medium only; for background removal)) was diluted in culture medium, and after uniformly mixing the drugs, the drugs were added to the corresponding cell wells and incubated in a CO2 incubator (37°C) for 3 days. After 3 days, 20 µl of MTS (Promegm, G3581) was added to each well and allowed to react for 2 hours. The readings were then taken at 490 nM using a microplate reader (Molecular Device, model: SpectraMAX190). The IC50 was calculated by detecting the activity of dehydrogenases in mitochondria to evaluate the inhibitory effect of camptothecin drugs on cell proliferation. The measurement results for the corresponding camptothecin chiral derivatives' IC50 (nM) are shown in the table below. [Table 5]

[0145] Dxd is the active ingredient of the commercially available anticancer ADC drug Enhertu and is a highly active camptothecin-based drug. Through cell activity experiments, the inventors have demonstrated that the camptothecin chiral derivative described in this invention exhibits cell activity equal to or greater than that of Dxd in representative tumor cells BXPC-3, A431, SW620, MDA-MB-468, and SK-BR-3.

[0146] Example 22: In vitro plasma stability of ADCs A specific amount of sterile ADC sample was taken and placed in sterile human plasma from which human IgG had been removed so that the final ADC concentration was 0.6 mg / ml. Each ADC was prepared in three tubes and incubated in a 37°C water bath for 0 h, 72 h, and 7 d hours, respectively. After incubation, the ADC sample was removed, 100 μL of Protein A (MabSelect SuRe TMLX Lot:#10221479GE, taken after washing with PBS) was added to each tube, and adsorption was allowed for 2 hours by agitation in a vertical mixer. After washing, elution, and Tris-HCl neutralization steps were performed to obtain incubated ADC. The DAR value of the ADC sample incubated for a specific time was measured by RP-HPLC to determine the plasma stability of the sample. As can be seen from the experimental results, the ADC of the present invention is hardly lost or loses very little during the plasma incubation process and has good stability in plasma. The possibility of toxicity due to premature degradation is low.

[0147] [Table 6]

[0148] Example 23: ADC antitumor cell activity test In the present invention, MDA-MB-468 and BT474 are used as research systems for in vitro efficacy detection. An appropriate amount of the tumor cell line was uniformly inoculated into a 96-well plate and incubated in a CO2 incubator. After 24 hours, after confirming that the cell state was normal under a microscope, drugs were added for treatment. The drug (the initial concentration of the ADC drug was 500 nM, the dilution factor was 7-fold, there were a total of 8 concentration points, the theoretical coupling ratio (DAR) of the toxin and the antibody was 8:1, and the actual coupling ratio was about 7.5:1. Therefore, the initial concentration of the toxin was 4.0 μM, diluted with a 7-fold concentration gradient, and there were 8 concentration points) was diluted in the medium, uniformly mixed, and then added to the corresponding cell wells. The next 2 columns were the control group (i.e., cells + medium, without drug treatment) and the blank group (i.e., without cells, containing only medium, for background removal), respectively. It was incubated in a CO2 incubator (37 °C) for 5 days. After 5 days, 20 μL of MTS (Promega, G3581) was added to each well and reacted for 2 hours, and the absorbance value at a wavelength of 490 nm was read using a microplate reader (Molecular Device, model number: SpectraMAX190). The inhibitory effect of the ADC drug on the proliferation of tumor cells was evaluated by calculating the IC50 by detecting the activity of dehydrogenase in mitochondria.

[0149]

Table 7

[0150] ADC-1 is the commercially available anti-tumor drug Enhertu. As can be seen from the above ADC cell activity test, the camptothecin chiral derivative drug described in the present invention shows good anti-tumor activity in many tumor cell lines after being coupled to an antibody via the binding unit L. Compared with ADC-1, some ADC activities are more excellent and the clinical application value is very high.

[0151] Example 24: ADC In Vivo Pharmacodynamic Experiment In this invention, a single-tumor NCI-N87 tumor-carrying nude mouse model was constructed to evaluate the in vivo efficacy of ADC coupling drugs. Specifically, 5 × 10⁻⁶ 6 NCI-N87 cells were subcutaneously inoculated into the right shoulder of 4-6 week old BALB / c nude mice, and the average volume of the mouse tumors was 162 mm². 3 After this stage, the mice were randomly divided into groups of 5 based on tumor size. On days 0, 7, 14, and 21, they received intravenous injections of a blank control (buffer blank) and antibody-drug conjugates ADC-1, ADC-2, ADC-3, ADC-5, ADC-6, and ADC-7 at a dose of 3 mg / kg, respectively. Tumor volume was measured as the mean tumor volume at the time of measurement. Changes in mouse body weight were recorded to observe the in vivo efficacy and initial toxicity of the ADC drugs.

[0152] [Table 8]

[0153] ADC-1 is the commercially available antitumor drug Enhertu. In the above ADC mouse in vivo efficacy experiments, the camptothesin chiral derivative drugs described in this invention, after being coupled to an antibody via binding unit L, exhibited clear antitumor activity in tumor-bearing mice, demonstrating a significantly smaller mean tumor volume compared to the blank control. Compared to ADC-1, the activity of ADC-2 was at the same level, ADC-5 was higher, and ADC-7 showed superior activity. Since there was no significant change in mouse body weight during the treatment period and no mouse deaths within the group, the camptothesin chiral derivative drugs described in this invention have good safety and high research application value.

Claims

1. Below formula D １ Camptothecin derivatives having the structure represented by , tautomers thereof, racemates, enantiomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof. 【Transformation 3】 (In the formula, 【Chemistry 4】 The chiral carbon atom bonded to it has an R absolute configuration. R is C １－３ Selected from alkyl groups, X is selected from -C(O)-CR ａ R ｂ -(CR ３ R ４ ) ｍ -O-, -C(O)-CR ａ R ｂ -(CR ３ R ４ ) ｍ -NH- or -C(O)-CR ａ R ｂ -(CR ３ R ４ ) ｍ -S-, and R ａ This is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls. R ｂ is selected from hydrogen atoms, deuterium atoms, halogens, alkyls, deuterated alkyls, alkyl halides, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclyls, aryls, substituted aryls, or heteroaryls, or R ａ , R ｂ And the carbon atoms bonded to them are C ３－６ Constituting a cycloalkyl or heterocycline, R ３ , R ４ These may be the same or different, and each is independently a hydrogen atom, a deuterium atom, a halogen, an alkyl, an alkyl halide, an alkyl deuterated, an alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyalkyl, a cycloalkyl, or a heterocyclyl, or R ３ , R ４ And the carbon atoms bonded to them are C ３－６ Constituting a cycloalkyl or heterocycline, m is selected from integers between 0 and 4.

2. A ligand-drug conjugate, 【Chemistry 15】 【change】 【change】 【change】 Selected from, where Ab is a ligand unit and n is selected from an integer or decimal number between 1 and 20. A ligand-drug complex having either an R absolute configuration or an S absolute configuration at position 1, or a pharmaceutically acceptable salt or solvate thereof, a tautomer thereof, a racemic mixture thereof, an enantiomer, or a mixture thereof.

3. The pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts formed with the acidic functional group in the structural formula; or acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbicates, salicylates, formates, glutamates, mesylates, ethanesulfons, benzenesulfons or p-toluenesulfons formed with the basic functional group in the structure, as described in claim 1, the camptothecin derivatives therein, tautomers, racemates, enantiomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, as described in claim 2, or pharmaceutically acceptable salts or solvates thereof, tautomers, racemates, enantiomers or mixtures thereof.

4. A pharmaceutical composition comprising a camptothecin derivative according to claim 1, its tautomer, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a ligand-drug conjugate according to claim 2, or a pharmaceutically acceptable salt or solvate thereof, its tautomer, racemate, enantiomer, or mixture thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

5. A pharmaceutical composition for treating or preventing tumors, comprising: a camptothecin derivative according to claim 1, a tautomer, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof; or a ligand-drug conjugate according to claim 2, or a pharmaceutically acceptable salt or solvate thereof, a tautomer, racemate, enantiomer, or mixture thereof; and a pharmaceutically acceptable carrier, diluent, or excipient.

6. The pharmaceutical composition according to claim 5, wherein the tumor is a solid tumor and a hematological tumor.

7. The pharmaceutical composition according to claim 5, wherein the tumor is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, or leukemia.

Images