Multifunctional cell membrane anchoring molecule and application thereof

Abstract

The invention relates to a compound shown in a general formula I or a stereoisomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof. The compound can be anchored to the surface of a living cell through a hydrophobic acting force between a hydrophobic tail chain and a cell membrane and a receptor-ligand acting force between a ligand branch chain and cell membrane protein, the anchoring efficiency is high, the anchoring stability is high, and the treatment effect of the living cell on diseases can be enhanced; meanwhile, a biological orthogonal group can be introduced into the surface of a cell, and the cell anchored by the compound can be coupled with a nano-drug, a protein drug or a fluorescent probe and the like through a biological orthogonal reaction, so that the treatment effect on diseases can be remarkably improved, and in-vivo and in-vitro tracing of living cells can be realized. The compound shown in the general formula I provides a new technical platform for cell-based immune combination therapy, and provides a new thought and a new medicine for cell therapy based on various diseases. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a multifunctional cell membrane anchoring molecule and application thereof. Background Art

[0002] Cancer, also known as malignant tumor, is the main cause of death. In addition to traditional therapies such as surgery, chemotherapy, and radiotherapy, cancer immunotherapy can exert anti-tumor effects by activating the body's own immune system. As the third revolution in tumor treatment, it has attracted much attention. At present, there are many tumor immunotherapies, mainly including immune checkpoint blockade (ICB), co-stimulatory receptor agonists, lymphocyte activation factors, oncolytic viruses, cancer vaccines, and cell immunotherapy. Among them, adoptive T cell therapy, including chimeric antigen receptor T cells (CAR-T cells), T cell receptor gene engineered T cells (TCR-T cells), and tumor infiltrating T lymphocytes (TIL), has attracted widespread attention due to its superior therapeutic effect; currently, 6 CAR-T cells have been approved by the FDA for marketing, all of which are used to treat hematological tumors, with significant efficacy and even the possibility of cure.

[0003] Although adoptive T cell therapy has achieved good results in patients with hematological tumors, factors such as the decline in tumor homing of immune cells, immunosuppressive microenvironment (TME), heterogeneous expression of tumor antigens, limited angiogenesis and hypoxia have limited the application of adoptive T cell therapy in solid tumors. Among them, the tumor immunosuppressive microenvironment can significantly inhibit the activity of tumor-infiltrating immune cells, which is the key to limiting the therapeutic effect of adoptive T cells on solid tumors.

[0004] Studies have shown that various immunosuppressive molecules in the tumor microenvironment, such as adenosine (ADO), lactic acid, TGF-β, etc., directly or indirectly inhibit T cell proliferation and secretion of effector factors after interacting with receptors on the T cell membrane, thereby reducing the activity of T cells. The combination of adoptive T cells and immunosuppressive molecule blockers can significantly improve the anti-tumor effect of overexcited T cells without producing obvious toxic side effects. However, this combined drug administration strategy is relatively complicated and patient compliance is poor; and the time point of administration is difficult to control, and the separate administration of drugs leads to inconsistent time points of action, which cannot achieve the best therapeutic effect. The Chinese patent "ZL202010815772.6" relates to a method for anchoring modified nanoparticles on the surface of living cells, and discloses a class of cell membrane anchoring molecules. However, this method has certain defects: the anchoring efficiency and long-term stability of anchoring molecules on the cell membrane are poor. Therefore, the development of a new method for anchoring and modifying the surface of living cells is of great significance and has broad application prospects.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The present disclosure provides a compound represented by general formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts:

[0007]

[0008] in:

[0009] R 1 Selected from distearoylphosphatidylethanolamine (DSPE), 1,2-hexadecanoylphosphatidylethanolamine (DPPE), 1,2-tetradecanoylphosphatidylethanolamine (DMPE), 1,2-dioleoyl-SN-glycero-3-phosphatidylethanolamine (DOPE), cholesterol (Cholesterol), tocopherol (Tocopherol) and

[0010] R 2 Selected from H, D, halogen and C 1-30 alkyl;

[0011] X is selected from azido, C 2-6 Alkynyl,

[0012] Y is selected from a bond, O, S, carbonyl, C 1-6 Alkylene, imino, sulfone and sulfoxide groups, the C 1-6 Alkylene and imino are represented by one or more R Y replaced by;

[0013] R Yare each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl;

[0014] Z is selected from C 1-6 alkyl, The C 1-6 The alkyl group is replaced by one or more R Z replaced by;

[0015] R Z are each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl;

[0016] n is any integer from 0 to 45;

[0017] m is any integer from 25 to 120;

[0018] p is 1, 2, 3, 4 or 5;

[0019] q is 1, 2, 3 or 4.

[0020] Effects of the Invention

[0021] The present invention provides a multifunctional cell membrane anchoring molecule shown in general formula I, wherein the anchoring molecule is composed of three parts: bialkane chains of different types and lengths as hydrophobic tail chains, polyethylene glycol (PEG) chains containing living cell membrane protein ligands as ligand side chains, and PEG chains containing bioorthogonal active groups as reaction main chains. The anchoring molecule can be anchored to the surface of living cells through the hydrophobic force between the hydrophobic tail chain and the cell membrane and the receptor-ligand force between the ligand side chains and the cell membrane protein, with high anchoring efficiency and strong anchoring stability, and can enhance the therapeutic effect of living cells on diseases.

[0022] In addition, the multifunctional cell membrane anchoring molecule shown in general formula I can introduce the bioorthogonal active group at the end of the reaction main chain to the surface of living cells. The living cell membrane anchoring technology developed by using it has the advantages of being simple and fast. The cells prepared by the living cell anchoring technology can be used in combination with the loaded drugs to significantly improve the therapeutic effect on the disease. The multifunctional cell membrane anchoring molecule shown in general formula I of the present invention can provide a new technical platform for cell-based immune combined therapy, and provide new ideas and new drugs for cell therapy based on various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention demonstrates a process in which the multifunctional cell membrane anchoring molecule shown in Formula I of the present application is anchored to the surface of living cells through the hydrophobic force between the hydrophobic tail chain and the cell membrane and the receptor-ligand force between the ligand side chain and the cell membrane protein, and the bioorthogonal active group is introduced into the surface of living cells.

[0024] Figure 2 The toxicity evaluation of the multifunctional anchoring molecules in Examples 1 to 4 on T cells is shown.

[0025] Figure 3 These are laser confocal images of the anchoring molecules in Examples 1 to 4 anchoring T cells.

[0026] Figure 4 The anchoring efficiency of the anchoring molecules in Examples 1 to 4 on T cells is shown (flow cytometry).

[0027] Figure 5 The anchoring stability of the anchor molecules in Examples 1 to 4 to T cells was shown (flow cytometry).

[0028] Figure 6 The anchor molecules in Examples 1 to 4 have the ability to enhance the activity of T cells. DETAILED DESCRIPTION

[0029] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0030] The present disclosure provides a compound represented by general formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts:

[0031]

[0032] in:

[0033] R 1 Selected from distearoylphosphatidylethanolamine (DSPE), 1,2-hexadecanoylphosphatidylethanolamine (DPPE), 1,2-tetradecanoylphosphatidylethanolamine (DMPE), 1,2-dioleoyl-SN-glycero-3-phosphatidylethanolamine (DOPE), cholesterol (Cholesterol), tocopherol (Tocopherol) and

[0034] R 2 Selected from H, D, halogen and C 1-30 alkyl;

[0035] X is selected from azido, C 2-6 Alkynyl,

[0036] Y is selected from a bond, O, S, carbonyl, C1-6 Alkylene, imino, sulfone and sulfoxide groups, the C 1-6 Alkylene and imino are represented by one or more R Y replaced by;

[0037] R Y are each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl;

[0038] Z is selected from C 1-6 alkyl, The C 1-6 The alkyl group is replaced by one or more R Z replaced by;

[0039] R Z are each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl;

[0040] n is any integer from 0 to 45;

[0041] m is any integer from 25 to 120;

[0042] p is 1, 2, 3, 4 or 5;

[0043] q is 1, 2, 3 or 4.

[0044] In certain embodiments, the R 2 Selected from H, D, halogen and C 9-21 alkyl.

[0045] In certain embodiments, the R 2 Selected from H, D, halogen and C 14-18 alkyl.

[0046] In certain embodiments, the R 2 Selected from C 14-18 alkyl.

[0047] In certain embodiments, the R 2 Selected from tetradecyl, hexadecyl and octadecyl.

[0048] In certain embodiments, q is 2.

[0049] In certain embodiments, the R 1 Selected from distearoylphosphatidylethanolamine (DSPE), 1,2-hexadecanoylphosphatidylethanolamine (DPPE), 1,2-tetradecanoylphosphatidylethanolamine (DMPE),

[0050] In certain embodiments, X is selected from azido, C 2-4 Alkynyl,

[0051] In certain embodiments, X is selected from azido, ethynyl,

[0052] In certain embodiments, said X is selected from azido.

[0053] In certain embodiments, Y is selected from a bond, O, S, carbonyl, C 1-3 Alkylene, imino, sulfone and sulfoxide groups, the C 1-3 Alkylene and imino are represented by one or more R Y replaced.

[0054] In certain embodiments, Y is selected from an imino group, wherein the imino group is replaced by one or more R Y replaced.

[0055] In certain embodiments, the R Y are each independently selected from H, D, halogen, amino, hydroxyl and C 1-3 alkyl.

[0056] In certain embodiments, the R Y are each independently selected from H.

[0057] In certain embodiments, Z is selected from C 1-3 alkyl, The C 1-3 The alkyl group is replaced by one or more R Z replaced.

[0058] In certain embodiments, Z is selected from methyl, The methyl group is replaced by one or more R Z replaced.

[0059] In certain embodiments, Z is selected from

[0060] In certain embodiments, the R Z are each independently selected from H, D, halogen, amino, hydroxyl and C 1-3 alkyl.

[0061] In certain embodiments, the R Z are each independently selected from H.

[0062] In some embodiments, n is any integer between 10 and 30.

[0063] In some embodiments, n is any integer between 20 and 25.

[0064] In certain embodiments, n is 23.

[0065] In some embodiments, m is any integer between 30 and 60.

[0066] In some embodiments, m is any integer between 40 and 50.

[0067] In certain embodiments, m is 46.

[0068] In certain embodiments, p is 4.

[0069] The present disclosure provides a compound or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, characterized in that the compound is selected from:

[0070]

[0071] In the present invention, the compound shown in the general formula I is composed of three parts: a bialkane chain of different types and lengths as a hydrophobic tail chain, a polyethylene glycol (PEG) chain containing a living cell membrane protein ligand as a ligand side chain, and a PEG chain containing a bio-orthogonal active group (i.e., the X) as a reaction main chain. It can be anchored to the surface of living cells through the hydrophobic force between the hydrophobic tail chain and the cell membrane and the receptor-ligand force between the ligand side chain and the living cell membrane protein, thereby enhancing the therapeutic effect of living cells such as adoptive T cells on diseases. In addition, by introducing the bio-orthogonal active group at the end of the reaction main chain of the compound shown in the general formula I into the surface of living cells, nanomedicines, proteins, fluorescent probes, etc. can be modified on the surface of living cells, providing new technologies and strategies for cell-based combined immunotherapy and cell in vivo tracing.

[0072] The present disclosure also provides a cell, which comprises the aforementioned compound or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt.

[0073] In certain embodiments, the cell is a cell having the aforementioned compound or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative, and pharmaceutically acceptable salt thereof anchored on its surface.

[0074] In certain embodiments, the cell is selected from primary cells or immortalized cells of humans or animals having a lipid membrane structure.

[0075] In certain embodiments, the cell is selected from the group consisting of a tumor cell, a neutrophil, a T cell, a mesenchymal stem cell, a hematopoietic stem cell, a natural killer cell, an antigen presenting cell, and a macrophage.

[0076] In certain embodiments, the cell is selected from a T cell and a neutrophil.

[0077] In certain embodiments, the cell is selected from a T cell.

[0078] In certain embodiments, the T cells are selected from chimeric antigen receptor T cells, T cell receptor genetically engineered T cells, and ordinary unmodified T cells.

[0079] In certain embodiments, the cell is a living cell.

[0080] The present invention also provides a method for anchoring and modifying cells, which comprises the step of co-incubating the aforementioned compound or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts with cells.

[0081] In certain embodiments, the concentration range of the aforementioned compound or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt is selected from 0.01 μM to 120 μM.

[0082] In certain embodiments, the concentration range of the aforementioned compound or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt is selected from 2 μM to 80 μM.

[0083] In certain embodiments, the co-incubation temperature is selected from 0°C to 40°C.

[0084] In certain embodiments, the co-incubation time is selected from 1 min to 60 min.

[0085] In certain embodiments, the cell is selected from primary cells or immortalized cells of humans or animals having a lipid membrane structure.

[0086] In certain embodiments, the cell is selected from the group consisting of a tumor cell, a neutrophil, a T cell, a mesenchymal stem cell, a hematopoietic stem cell, a natural killer cell, an antigen presenting cell, and a macrophage.

[0087] In certain embodiments, the cell is selected from a T cell and a neutrophil.

[0088] In certain embodiments, the cell is selected from a T cell.

[0089] In certain embodiments, the T cells are selected from chimeric antigen receptor T cells, T cell receptor genetically engineered T cells, and ordinary unmodified T cells.

[0090] In certain embodiments, the cell is a living cell.

[0091] In the present invention, for cells with multifunctional anchoring molecules (i.e., the aforementioned compounds or their stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts) anchored on the surface, the activity of living cells, especially immune cells, is increased due to the receptor-ligand interaction between the ligand side chains in the anchoring molecule structure and the inhibitory membrane proteins of living cells. The survival rate of living cells is >80%, and the normal physiological functions of living cells, including cell survival rate, chemotaxis to diseases, etc., are maintained.

[0092] The present disclosure also provides a cell medicine, which comprises the aforementioned cells and bioactive molecules.

[0093] In certain embodiments, the bioactive molecule is selected from one or more of a fluorescent probe, a protein, and a nanomedicine.

[0094] In certain embodiments, the fluorescent probe is selected from one or both of a fluorescent dye and an AIE probe.

[0095] In certain embodiments, the fluorescent dye is selected from one or more of FITC, DIR and DIL.

[0096] In certain embodiments, the protein is selected from one or both of a therapeutic antibody and a chemokine.

[0097] In certain embodiments, the therapeutic antibody is selected from one or both of PD-1 monoclonal antibody and PD-L1 monoclonal antibody.

[0098] In certain embodiments, the chemokine is selected from CXCL-8.

[0099] In certain embodiments, the nanomedicine is selected from nanoparticles loaded with a therapeutic agent.

[0100] In certain embodiments, the nanoparticles are liposomes, nanovesicles, solid lipid nanoparticles or micelles with a particle size of 1 nm to 1000 nm.

[0101] In certain embodiments, the nanoparticles are liposomes, nanovesicles, solid lipid nanoparticles or micelles with a particle size of 10 nm to 500 nm.

[0102] In certain embodiments, the therapeutic agent is selected from one or more of avasimibe, paclitaxel, and PD-1 monoclonal antibody.

[0103] In certain embodiments, the drug loading of the therapeutic agent is 0.1% to 20%.

[0104] In certain embodiments, the drug loading of the therapeutic agent is 1% to 15%.

[0105] The present invention also provides a method for preparing the aforementioned cell medicine, comprising co-incubating the aforementioned cells and the aforementioned bioactive molecules, anchoring and modifying the bioactive molecules to the cell surface through a bioorthogonal click chemistry reaction between the bioorthogonal groups of the anchoring molecules on the cell membrane and the corresponding reactive groups on the bioactive molecules, and obtaining the aforementioned cell medicine.

[0106] In certain embodiments, the bioorthogonal click chemistry reaction includes ketone / hydroxylamine condensation, Michael addition reaction of sulfhydryl or amino group with maleimide, ring strain driven azide-alkyne cycloaddition reaction (SPAAC), high strain driven inverse electron demand Dields-Alder cycloaddition reaction (SPIEDAC).

[0107] In certain embodiments, during the co-incubation, the concentration of the cells ranges from 10 μg / mL to 200 μg / mL.

[0108] In certain embodiments, during the co-incubation process, the concentration of the bioactive molecule ranges from 5 μg / mL to 200 μg / mL.

[0109] In certain embodiments, the co-incubation time is 10 min to 60 min.

[0110] In certain embodiments, the co-incubation temperature is 4°C to 37°C.

[0111] In the present invention, the survival rate of living cells in the cell medicine is greater than 80%, and the normal physiological functions of living cells are maintained, including cell survival rate, chemotaxis to diseases, etc.

[0112] The present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the aforementioned compound, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or a therapeutically effective amount of the aforementioned cells or a therapeutically effective amount of the aforementioned cell drug.

[0113] In certain embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0114] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned compound based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 5%-95% of the aforementioned compound.

[0115] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned cells based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned cells. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned cells. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned cells. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned cells. In certain embodiments, the pharmaceutical composition contains 5%-95% of the aforementioned cells.

[0116] In certain embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned cell drug. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned cell drug. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned cell drug. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned cell drug. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned cell drug. In certain embodiments, the pharmaceutical composition contains 5%-95% of the aforementioned cell drug.

[0117] All compounds, compositions containing compounds of the present application, cells containing compounds of the present application, cell drugs containing cells of the present application, compositions containing cells of the present application, compositions containing cell drugs of the present application, etc., involved in the present application can be administered to the organism via any administration route. The administration route can be oral administration, intravenous injection, intramuscular injection, subcutaneous injection, intratumor injection, rectal administration, vaginal administration, sublingual infusion, nasal inhalation, oral inhalation, eye drops, and can also be local or systemic transdermal administration.

[0118] Different dosages can be selected according to the nature and intensity of the disease suffered by different individuals, the age, gender, weight of the patient, the route of administration and other factors. The dosage of the compound of the present application can be 0.01 to 500 mg / kg per day, preferably 1-100 mg / kg per day, and can be administered once or multiple times.

[0119] Different dosages can be selected according to the nature and intensity of the disease suffered by different individuals, the age, gender, weight of the patient, the route of administration and other factors. The dosage of the cells of the present application can be 1*10 per day. 5 cells / kg to 5*10 7 cells / kg, preferably 5*10 5 cells / kg to 1*10 7 cells / kg, can be administered in single or multiple doses.

[0120] Different dosages can be selected according to the nature and intensity of the disease suffered by different individuals, the age, gender, weight of the patient, the route of administration and other factors. The dosage of the cell drug of the present application can be 1*10 per day. 5 cells / kg to 5*10 7 cells / kg, preferably 5*10 5 cells / kg, can be administered in single or multiple doses (10 5 cells can be loaded with 0.01 to 100 mg of drug).

[0121] The present disclosure also provides the aforementioned compound, or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, or the aforementioned cell, or the aforementioned cell drug or the aforementioned pharmaceutical composition, which is used for preventing and / or treating diseases.

[0122] In certain embodiments, the disease is selected from a tumor and an autoimmune disease.

[0123] In certain embodiments, the tumor comprises melanoma, glioma, lung cancer, breast cancer, and ovarian cancer.

[0124] In certain embodiments, the autoimmune disease includes stroke, arthritis, and systemic lupus erythematosus.

[0125] The present disclosure also provides the use of the aforementioned compound, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the aforementioned cells, or the aforementioned cell drugs or the aforementioned pharmaceutical compositions for preventing and / or treating diseases.

[0126] In certain embodiments, the disease is selected from a tumor and an autoimmune disease.

[0127] In certain embodiments, the tumor comprises melanoma, glioma, lung cancer, breast cancer, and ovarian cancer.

[0128] In certain embodiments, the autoimmune disease includes stroke, arthritis, and systemic lupus erythematosus.

[0129] The present disclosure also provides the use of the aforementioned compound, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the aforementioned cells, or the aforementioned cell drugs or the aforementioned pharmaceutical compositions in the preparation of drugs for preventing and / or treating diseases.

[0130] In certain embodiments, the disease is selected from a tumor and an autoimmune disease.

[0131] In certain embodiments, the tumor comprises melanoma, glioma, lung cancer, breast cancer, and ovarian cancer.

[0132] In certain embodiments, the autoimmune disease includes stroke, arthritis, and systemic lupus erythematosus.

[0133] The present disclosure also provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned compound, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or a therapeutically effective amount of the aforementioned cells, or a therapeutically effective amount of the aforementioned cell drug, or a therapeutically effective amount of the aforementioned composition.

[0134] In certain embodiments, the disease is selected from a tumor and an autoimmune disease.

[0135] In certain embodiments, the tumor comprises melanoma, glioma, lung cancer, breast cancer, and ovarian cancer.

[0136] In certain embodiments, the autoimmune disease includes stroke, arthritis, and systemic lupus erythematosus.

[0137] The present disclosure also provides the use of the aforementioned compound, or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, or the aforementioned cell, or the aforementioned cell drug or the aforementioned pharmaceutical composition in in vivo visualization of cells.

[0138] The present disclosure also provides the use of the aforementioned compound, or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, or the aforementioned cell, or the aforementioned cell drug or the aforementioned pharmaceutical composition in the preparation of an agent for in vivo visualization of cells.

[0139] Terminology explanation:

[0140] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0141] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkyl group containing 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, 5-methylhexyl, 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 various branched chain isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which 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, and the like. The alkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably independently selected from one or more substituents of D atoms, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0142] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group, which is a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-、1,1-propylene(-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), etc. The alkylene group may be substituted or unsubstituted, and when substituted, it may be substituted at any available attachment point, and the substituents are preferably independently and optionally selected from one or more substituents of alkenyl, alkynyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0143] The term "alkenyl" refers to an alkyl compound containing at least one carbon-carbon double bond in the molecule, wherein the definition of alkyl is as described above. Alkenyl can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from one or more substituents in alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0144] The term "alkynyl" refers to an alkyl compound containing at least one carbon-carbon triple bond in the molecule, wherein the definition of alkyl is as described above. Alkynyl can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from one or more substituents in alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0145] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0146] The term "hydroxy" refers to -OH.

[0147] The term "amino" refers to -NH 2 .

[0148] The term "imino" refers to -NH-.

[0149] The term "sulfone" refers to -SO 2 -.

[0150] The term "sulfoxide" refers to -SO-.

[0151] The term "carbonyl" refers to C=O.

[0152] The term "azido" refers to an -N 3 .

[0153] In the chemical structure of the compound described in the present invention, the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both Two configurations. Key Can be Although all the above structural formulas are drawn as certain isomers for the sake of simplicity, the present invention may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds disclosed in the present invention, the bonds No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0154] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0155] "Substituted" means that one or more hydrogen atoms, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently by a corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0156] The term "isotopic derivative" refers to a compound whose structure differs only in the presence of one or more isotopically enriched atoms. For example, a compound having a structure disclosed herein with "deuterium" or "tritium" replacing hydrogen, or with 18 F-fluorine labeling ( 18 F isotope) instead of fluorine, or with 11 C-, 13 C-, or 14 C-enriched carbon ( 11 C-, 13 C-, or 14 C-carbon labeling; 11 C-, 13 C-, or 14 Compounds in which a carbon atom is replaced by a C-isotope) are within the scope of the present disclosure. Such compounds can be used as analytical tools or probes in, for example, biological assays, or can be used as in vivo diagnostic imaging tracers for diseases, or as tracers for pharmacodynamics, pharmacokinetics or receptor studies. The various deuterated forms of the compounds disclosed herein refer to compounds in which each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds with reference to relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds, or they can be synthesized using deuterated reagents using conventional techniques, and deuterated reagents include but are not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc. Deuterated substances can generally retain activity comparable to undeuterated compounds, and when deuterated at certain specific sites, better metabolic stability can be achieved, thereby obtaining certain therapeutic advantages.

[0157] The term "solvate" refers to a physical association of a compound of the present disclosure with one or more, preferably 1-3, solvent molecules, whether organic or inorganic. The physical association includes hydrogen bonding. In some cases, for example, when one or more, preferably 1-3, solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be isolated. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0158] The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water.

[0159] The term "prodrug" refers to a compound that can be transformed in vivo under physiological conditions, for example by hydrolysis in the blood, to yield the active prodrug.

[0160] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with patient tissues without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and effective for the intended use.

[0161] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the disclosed compounds that are safe and effective for use in mammals and have the desired biological activity. Salts can be prepared separately during the final isolation and purification of the compound, or by reacting a suitable group with a suitable base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases, as well as organic bases. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids and organic acids.

[0162] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0163] The term "cellular medicine" refers to cells that can be used to treat diseases, and the cells exert therapeutic effects on the diseases through the functions of the cells.

[0164] The term "nanomedicine" refers to the nanoscale preparation of drugs. Generally, it is called nanoparticles or nanocarriers or nanomedicines in pharmacy, and its size is defined between 1-1000nm. Among them, nanocarriers refer to various nanoparticles with drugs dissolved or dispersed. Nanomedicine refers to the direct processing of raw drugs into nanoparticles.

[0165] The term "nanoparticle" refers to particles with a particle size of the order of nanometers (nm). Nanoparticles generally refer to particles with a particle size of 1 nm to 1000 nm. Specific examples of nanoparticles include polymer nanoparticles, metal nanoparticles, dendrimers, and the like.

[0166] The term "liposome" refers to a vesicle containing a lipid membrane and an aqueous medium encapsulated in the lipid membrane. The lipid membrane of the liposome is composed of one or more lipid layers. For example, it is composed of a lipid bilayer containing phospholipids.

[0167] The term "micelle" refers to a vesicle formed by a monolayer molecular membrane. Examples of micelle components include amphiphilic molecules such as surfactants.

[0168] The term "incubation" refers to growing in a cell culture incubator.

[0169] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts thereof, or one or more cells described herein or one or more cell drugs described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0170] With respect to a drug or pharmacologically active agent, the term "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but can achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate effective amount in each case can be determined by a person skilled in the art based on routine experiments.

[0171] As used herein, the singular form of "a," "an," and "the" include plural references and vice versa unless the context clearly dictates otherwise.

[0172] In the claims and description of the present invention, unless the context requires otherwise due to expressive language or necessary implication, the word "comprises / includes" or variations such as "comprises / includes" or "includes / includes" are used in an inclusive sense, i.e., specifying the presence of the described features, but not excluding the presence or addition of other features in various embodiments of the present invention.

[0173] When the term "about" is applied to a parameter such as pH, concentration, temperature, etc., it indicates that the parameter can vary by ±10%, and sometimes more preferably within ±5%. As will be understood by those skilled in the art, when a parameter is not critical, numbers are generally given only for illustrative purposes and not for limitation.

[0174] The "room temperature" is not a specific temperature value, but refers to the temperature range of 10-30°C.

[0175] The compounds disclosed herein can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from easily prepared intermediates, by using standard synthetic methods and procedures known to those skilled in the art or apparent to the skilled person based on the teachings herein. Standard synthetic methods and procedures for preparing organic molecules and functional group transformations and manipulations can be obtained from relevant scientific literature or from standard textbooks in the art. Although not limited to any one or several sources, the classic textbooks incorporated herein by reference are Smith, MB, March, J., March'Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John.

[0176] The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for preparing compounds of the present disclosure. The compounds of the present invention having the various formulae described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature procedures according to the procedures described in the following representative synthetic methods. The variables (e.g., R 1 and R 2 The present invention relates to a kind of molecule. For example, the molecule of the present invention is a molecule of the present invention ...

[0177] The terms involved in the present invention are defined above. Those skilled in the art can also understand the above terms in combination with the prior art. The following is a further description based on the content of the present invention and the definitions of the terms.

[0178] The method of the present invention is described below by means of specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following examples; the implementation conditions adopted in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments; the reagents without specifying the specific source are conventional reagents purchased on the market.

[0179] Embodiment 1: Synthesis

[0180] Step 1: N 6 -(Azide-Polyethylene glycol 2000-acetyl)-N 2 Synthesis of -(tert-butyloxycarbonyl)-L-lysine

[0181]

[0182] N 2 -(tert-Butyloxycarbonyl)-L-lysine (19 mg, 0.075 mmol) and NHS-PEG 2000 -N 3(100 mg, 0.05 mmol) was dissolved in 4 mL DMSO, and 15 μL triethylamine was added dropwise under stirring. After reacting at room temperature for 24 h, it was transferred to a dialysis bag with a molecular weight cutoff of 1000, dialyzed against DMSO for 2 days and pure water for 2 days, and freeze-dried to obtain a white solid powder as the product (70 mg, yield: 64%). 1 H NMR (300 Hz, CDCl 3 ): δ7.14(s,1H),5.25(d,J=7.8Hz,1H),4.30(d,J=6.9Hz,1H),3.98(s,2H),3.88(s,1H),3.65(s ,173H),3.40(t,J=5.0Hz,3H),3.31(s,2H),1.93–1.70(m,2H),1.69–1.50(m,2H),1.44(s,9H).

[0183] Step 2: Synthesis of 3-(((2-(2-((tert-butyloxycarbonyl)amino)-6-(2-(azido-polyethylene glycol 2000)-acetamido)hexanamido)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-distearate (DSPE-BocLys-PEG 2000 -N 3 )

[0184]

[0185] N 6 -(Azide-Polyethylene glycol 2000-acetyl)-N 2 -(tert-Butyloxycarbonyl)-L-lysine (30 mg, 0.01336 mmol) and PyBop (11 mg, 0.02004 mmol) were dissolved in 3 mL of chloroform, and 20 μL of DIPEA was added dropwise under stirring, and activated at room temperature for 3 h. DSPE (10.3 mg, 0.01871 mmol) was dissolved in 2 mL of chloroform, and the DSPE solution was added dropwise to the reaction solution, and reacted at 37°C for 24 h. After the reaction was complete, the solvent was removed under reduced pressure, 3 mL of DMSO was added to dissolve, and the mixture was transferred to a dialysis bag with a molecular weight cutoff of 2000, dialyzed with DMSO for 2 days, dialyzed with pure water for 2 days, and freeze-dried to obtain a white powder product (27 mg, yield: 68%). 1 H NMR (300 Hz, CDCl 3): δ5.23(s,1H),4.36(d,J=12.0Hz,1H),4.10(s,6H),3.65(s,189H),3.40(t,J=5.0Hz,4H),3.15–2 .91(m,4H),2.31(q,J=7.0Hz,4H),1.67–1.51(m,7H),1.44(s,9H),1.25(s,57H),0.91-0.85(t,6H).

[0186] Step 3: Synthesis of 3-(((2-(2-amino-6-(2-(azido-polyethylene glycol 2000)-acetamido)hexanamido)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-distearate (DSPE-Lys-PEG 2000 -N 3 )

[0187]

[0188] The compound DSPE-BocLys-PEG 2000 -N 3 (25 mg, 0.0083 mmol) was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added dropwise under stirring. The reaction was carried out at room temperature for 3 h. After the reaction was complete, the solvent was removed under reduced pressure to obtain a crude compound, which was directly used in subsequent reactions.

[0189] Step 4: Synthesis of 3-(((2-(2-(methyl-polyethylene glycol 1000-acetamido)-6-(2-(azido-polyethylene glycol 2000)-acetamido)hexanamido)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-distearate (DSPE-(M-PEG 1000 )Lys-PEG 2000 -N 3 )

[0190]

[0191] DSPE-Lys-PEG 2000 -N 3 (21 mg, 0.007 mmol) and M-PEG 1000-COOH (9 mg, 0.009 mmol) was used as the raw material, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH, 4 mg, 0.014 mmol) was added and dissolved in 3 mL DMSO, 20 μL N-methylimidazole (NMI) was added dropwise under stirring, and the mixture was reacted at room temperature for 24 h, and then transferred to a dialysis bag with a molecular weight cutoff of 3000, dialyzed with DMSO for 2 days, dialyzed with pure water for 2 days, and freeze-dried to obtain a white powder solid as the control compound DSPE-(M-PEG 1000 )Lys-PEG 2000 -N 3 (24 mg, yield: 89%). 1 H NMR (300 Hz, CDCl 3 ): δ5.23(s,1H),4.35(s,1H),4.14(m,J=28.3Hz,8H),3.65(s,253H),3.40(d,J=5.0Hz,3 H),3.38(s,3H),2.66(s,8H),2.31(d,J=7.2Hz,4H),1.25(s,65H),0.88(t,J=6.5Hz,6H).

[0192] Embodiment 2: Synthesis

[0193] Step 1: Synthesis of 3-(2-furan)-1H-1,2,4-triazole-5-amine

[0194]

[0195] Sodium methoxide (8.643g, 160.0mmol) and aminoguanidine hydrochloride (8.844g, 80.0mmol) were dissolved in 50mL methanol under ice-water bath. Methyl 2-furoate (5.044g, 40.0mmol) was dissolved in 15mL methanol. Then, the methanol solution of methyl 2-furoate was added dropwise to the methanol solution of sodium methoxide and aminoguanidine hydrochloride under ice-water bath, stirring while adding. After the addition was completed, the mixture was refluxed at 75°C overnight under argon protection. After the reaction was completed, the mixture was cooled to room temperature, the solid was removed by suction, and the filter cake was washed with methanol three times. After the filtrate was dried, a reddish brown viscous solid was obtained, which was dissolved by adding 5mL water. The pH was adjusted to 4 with 3N hydrochloric acid, and the color of the solution changed from wine red to light yellow. Saturated NaHCO 3 The pH was adjusted to neutral, and solids were precipitated after partial removal of water. After suction filtration, the solids were washed three times with a small amount of water, and the filter cake was collected and dried to obtain a grayish yellow solid product (3.120 g, yield: 52%). 1 H NMR (300 Hz, DMSO-d 6): δ12.27(s,1H),7.69(dd,J=1.8,0.9Hz,1H),6.71(dd,J=3.3,0.9Hz,1H),6.55(dd,J=3.3,1.8Hz,1H),6.08(s,2H).

[0196] Step 2: Synthesis of 2-(furan-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-7-amine

[0197]

[0198] 3-(2-Furan)-1H-1,2,4-triazole-5-amine (2.0 g, 13.3 mmol) was mixed with dimethyl N-cyanodithioimidate (1.948 g, 13.3 mmol) and reacted at 180°C for 2 h under argon protection. After the reaction was completed, the mixture was cooled to room temperature to obtain a yellow solid crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain a white solid product (1.030 g, yield: 31%). 1 H NMR (300 Hz, DMSO-d 6 ): δ8.88(d,J=56.9Hz,2H),7.94(dd,J=1.8,0.8Hz,1H),7.17(dd,J=3.4,0.8Hz,1H),6.72(dd,J=3.4,1.8Hz,1H),2.51(s,4H).

[0199] Step 3: Synthesis of 2-(furan-2-yl)-5-(methylsulfonyl)-[1,2,4]triazolo[1,5-Α][1,3,5]triazine-7-amine

[0200]

[0201] Dissolve 2-(furan-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-A][1,3,5]triazine-7-amine (500 mg, 2.0 mmol) and m-chloroperbenzoic acid (1.380 g, 6.8 mmol) in 24 mL of dichloromethane (DCM) and 15 mL of dichloromethane, respectively, and then add the m-chloroperbenzoic acid solution dropwise to the solution of 2-(furan-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-A][1,3,5]triazine-7-amine under an ice-water bath, stirring while adding. After the addition is completed, transfer to room temperature for reaction for 22 hours. After the reaction was complete, the solvent was removed under reduced pressure and at low temperature, 8 mL of ethanol was added, and the mixture was stirred at room temperature for 30 min. A gray-brown solid was precipitated, and the gray-brown solid was obtained by filtration. The filter cake was washed with a small amount of ethanol and dried to obtain (340 mg, yield: 61%).1 H NMR (300 Hz, DMSO-d 6 ): δ9.81(s,1H),9.48(s,1H),8.04–7.92(m,1H),7.27(dd,J=3.5,0.8Hz,1H),6.76(dd,J=3.5,1.8Hz,1H),3.36(s,3H).

[0202] Step 4: Synthesis of ethyl 2-(4-(2-(tert-butoxycarbonyl)amino)ethyl)phenoxy)acetate

[0203]

[0204] Dissolve tert-butyl 4-hydroxyphenethylcarbamate (2.374 g, 10 mmol) in 8 mL N,N-dimethylformamide (DMF), add anhydrous potassium carbonate (2.074 g, 15 mmol), 18-crown-6 (0.132 g, 0.5 mmol) and ethyl 2-bromoacetate (2.004 g, 12 mmol), and stir overnight at room temperature. After the reaction is completed, add 50 mL of ethyl acetate to dilute, wash the organic layer three times with water and saturated brine, dry over anhydrous sodium sulfate, and distill under reduced pressure to remove the solvent to obtain a colorless oily liquid. After cooling, a white waxy solid product (3.0 g, yield: 95%) is obtained. 1 H NMR (300 Hz, CDCl 3 ): δ7.14–7.07(m,2H),6.91–6.80(m,2H),4.60(s,2H),4.27(q,J=7.2Hz,2H),3 .34(q,J=6.8Hz,2H),2.73(t,J=7.0Hz,2H),1.43(s,9H),1.30(t,J=7.1Hz,3H).

[0205] Step 5: Synthesis of 2-(4-(2-(tert-butyloxycarbonyl)amino)ethyl)phenoxy)acetic acid

[0206]

[0207] Ethyl 2-(4-(2-(tert-butoxycarbonyl)amino)ethyl)phenoxy)acetate (3.0 g, 9.283 mmol) was dissolved in 20 mL of tetrahydrofuran / water mixed solution (1:1, v / v), 30 mL of 1N lithium hydroxide aqueous solution was added, and the mixture was reacted at room temperature for 3 h. After the reaction was completed, tetrahydrofuran was removed under reduced pressure, and saturated citric acid was added to adjust the pH to acidic. A large amount of white solid was produced, which was filtered and dried to obtain a white solid product (2.548 g, yield: 93%). 1 H NMR (300 Hz, CDCl 3): δ7.12(d,J=9.1Hz,2H),6.86(d,J=8.0Hz,2H),4.63(s,2H),3.33(s,2H),2.73(t,J=7.5Hz,2H),1.43(s,9H).

[0208] Step 6: Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(4-(2-((tert-butyloxycarbonyl)amino)ethyl)phenoxy)acetate

[0209]

[0210] 2-(4-(2-(tert-Butyloxycarbonyl)amino)ethyl)phenoxy)acetic acid (150 mg, 0.5079 mmol), N-hydroxysuccinimide (88 mg, 0.7618 mmol) and HATU (290 mg, 0.7618 mmol) were dissolved in 10 mL of dichloromethane, and 140 μL of N,N-diisopropylethylamine (DIPEA) was added while stirring, and the mixture was reacted at room temperature for 3 h. After the reaction, 20 mL of dichloromethane was added, and the organic layer was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude white solid obtained by column chromatography with dichloromethane / methanol (50:1, v / v) as the eluent was used directly in the subsequent reaction.

[0211] Step 7: Synthesis of 2-(4-(2-(tert-butyloxycarbonyl)amino)ethyl)phenoxy)acetyl-polyethylene glycol 1000-acetic acid

[0212]

[0213] The crude 2,5-dioxopyrrolidin-1-yl-2-(4-(2-((tert-butyloxycarbonyl)amino)ethyl)phenoxy)acetate was reacted with H 2 N-PEG 1000 -COOH (180 mg, 0.18 mmol) was dissolved in 3 mL of dichloromethane, and 150 μL of triethylamine (TEA) was added dropwise under stirring, and the mixture was reacted at room temperature for 12 h. After the reaction was completed, dichloromethane was removed by distillation under reduced pressure, and the mixture was dissolved in dimethyl sulfoxide (DMSO), and then transferred to a dialysis bag with a molecular weight cutoff of 500, dialyzed with DMSO for 2 days, and then dialyzed with pure water for 2 days. After freeze drying, a white solid powder was obtained as the product (100 mg, yield: 44%). 1 H NMR (300 Hz, CDCl 3): δ7.16–7.10(dd,2H),7.04(s,1H),6.91–6.83(dd,2H),4.67–4.56(m,1H),4.48(s,2H),4.16(s,2H),3.89(d,J=5.0Hz,1H),3.79–3.7 3(m,2H),3.71(s,3H),3.68–3.60(s,114H),3.59–3.53(m,5H),3.42(s,1H),3.33(d,J=6.7Hz,2H),2.74(t,J=7.1Hz,2H),1.44(s,9H).

[0214] Step 8: Synthesis of 2-(4-(2-aminoethyl)phenoxy)acetyl-polyethylene glycol 1000-acetic acid

[0215]

[0216] 2-(4-(2-(tert-Butyloxycarbonyl)amino)ethyl)phenoxy)acetyl-polyethylene glycol 1000-acetic acid (50 mg, 0.039 mmol) was dissolved in 1.2 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added dropwise under stirring. The reaction was carried out at room temperature for 6 h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude compound, which was directly used in subsequent reactions.

[0217] Step 9: 2-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenoxy)acetyl-polyethylene glycol 1000-acetic acid (ZM-PEG 1000 Synthesis of -COOH)

[0218]

[0219] The crude product of 2-(4-(2-aminoethyl)phenoxy)acetyl-polyethylene glycol 1000-acetic acid and 2-(furan-2-yl)-5-(methylsulfonyl)-[1,2,4]triazolo[1,5-Α][1,3,5]triazine-7-amine (23 mg, 0.08 mmol) were dissolved in 3 mL DMSO, and 50 μL triethylamine was added dropwise under stirring and reacted at room temperature for 24 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 500, dialyzed with DMSO for 2 days, dialyzed with pure water for 2 days, and the yellow-brown powder obtained by freeze drying was the product (32 mg, yield: 52%). 1 H NMR (300 Hz, CDCl 3): δ7.56(d,J=1.8Hz,1H),7.17(dd,J=13.0,5.8Hz,3H),6.84(d,J=8.3Hz,2H),6.55(dd,J=3.4,1.8 Hz,1H),4.48(s,2H),4.15(s,2H),3.75(s,3H),3.64(s,117H),3.47(s,2H),2.90(d,J=7.0Hz,2H).

[0220] Step 10: 3-(((2-(2-(2-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenoxy)acetyl-polyethylene glycol 1000-acetamido)-6-(2-((2-azidoethoxy)-polyethylene glycol 2000)-acetamido)hexanamido)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-distearate (DSPE-(ZM-PEG 1000 )Lys-PEG 2000 -N 3 )

[0221]

[0222] The detailed operation steps refer to step 4 in Example 1, using ZM-PEG 1000 -COOH replaces the raw material M-PEG 1000 -COOH, and finally a yellow-brown powder product was obtained. 1 H NMR (300 Hz, CDCl 3 ): δ7.57(s,1H),7.17(d,J=9.7Hz,3H),7.08(s,1H),6.87(d,J=7.9Hz,2H),6.55(s,1H),5.24(s,1H),4.49(s,1H),4.36(s,1H),4.07 (s,5H),4.00(s,1H),3.65(s,257H),3.40(d,J=4.9Hz,4H),2.89(s,3H),2.31(d,J=7.2Hz,4H),1.25(s,54H),0.88(t,J=6.6Hz,6H).

[0223] Embodiment 3: Synthesis

[0224] Step 1: Synthesis of L-glutamic acid dioctadecanol ester

[0225]

[0226] Glutamic acid (6 g, 40.78 mmol) and p-toluenesulfonic acid (7.76 g, 45.06 mmol) were dissolved in 250 mL of toluene, a water separator was installed, and the mixture was heated to reflux at 145°C for 3 h. After cooling to room temperature, 1-octadecanol (23.17 g, 85.66 mmol) was added, and the mixture was heated to reflux at 145°C for overnight reaction. After the reaction was complete, the insoluble matter was removed by filtration, the toluene was removed by vacuum, dichloromethane was added to dissolve the solid, and saturated NaHCO was added. 3 The p-toluenesulfonic acid was removed, and then the organic layer was washed three times with water and saturated NaCl, respectively, and the organic layer was collected and the solvent was removed under reduced pressure to obtain a crude product. The crude product was recrystallized from methanol to obtain the product (20.162 g, yield: 77%). 1 H NMR (300 Hz, CDCl 3 ): δ4.11(dt,J=15.4,6.8Hz,4H),3.55(dd,J=8.2,5.2Hz,1H),2.49(t,J=7.5Hz,2H),2.18 –2.05(m,1H),1.98–1.83(m,1H),1.64(m,J=6.9Hz,4H),1.27(s,63H),0.93–0.86(t,6H).

[0227] Step 2: N 6 -((9H-fluorene-9-methoxy)carbonyl)-N 2 Synthesis of -(tert-butyloxycarbonyl)-L-lysyl-L-glutamic acid bis(octadecyl) ester

[0228]

[0229] Compound L-glutamic acid dioctadecyl ester (6.521 g, 10 mmol), N 2 -tert-butyloxycarbonyl-N 6 -Fluorenylmethoxycarbonyl-L-lysine (4.920 g, 10.5 mmol) and 4-dimethylaminopyridine (147 mg, 1.2 mmol) were dissolved in 100 mL of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (2.3 g, 12 mmol) was added under stirring in an ice-water bath, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, a white solid product (5.71 g, yield: 51%) was obtained after purification by column chromatography. 1 H NMR (300 Hz, CDCl 3): δ7.76(dd,J=7.5Hz,2H),7.60(d,J=7.5Hz,2H),7.40(t,J=7.4Hz,2H),7.31(td,J=7.4,1.1Hz,2H),6.78(d,J=7.8Hz ,1H),5.07(d,J=36.7Hz,2H),4.59(td,J=8.0,4.8Hz,1H),4.39(d,J=6.8Hz,2H),4.21(t,J=7.0Hz,1H),4.15–4.08(m,4 H),4.05(t,J=6.8Hz,2H),3.21(d,J=6.6Hz,2H),2.38(m,J=16.7,12.3,6.9Hz,2H),2.21(tq,J=12.3,7.4,6.1Hz,1H), 2.02–1.94(m,1H),1.87(d,J=12.0Hz,1H),1.65–1.53(dq,6H),1.44(s,9H),1.32–1.22(m,62H),0.88(t,J=6.8Hz,6H).

[0230] Step 3: N 2 Synthesis of -(tert-butyloxycarbonyl)-L-lysyl-L-glutamic acid bis(octadecyl) ester

[0231]

[0232] N 6 -((9H-fluorene-9-methoxy)carbonyl)-N 2 -(tert-Butyloxycarbonyl)-L-lysyl-L-glutamic acid bis(octadecyl) ester (1.103 g, 1.0 mmol) was dissolved in 8 mL of dichloromethane, and 2 mL of diethylamine was added under stirring, and the reaction was allowed to proceed overnight at room temperature. After the solvent was removed under reduced pressure, 10 mL of ether was added, and the mixture was stirred at room temperature for 5 min. After filtration and drying, a light yellow solid product (610 mg, yield: 69%) was obtained. 1 H NMR (400 Hz, CDCl 3): δ5.25(d,J=8.0Hz,1H),4.57(td,J=8.0,5.0Hz,1H),4.16(d,J=7.1Hz,1H),4.11(t,J =6.8Hz,2H),4.05(t,J=6.8Hz,2H),2.84(t,J=7.0Hz,2H),2.40(m,J=10.3,9.6Hz,2H), 2.21(tt,J=13.6,6.1Hz,1H),2.01(ddd,J=12.2,8.5,6.3Hz,1H),1.83(dt,J=14.0,7.4 Hz,1H),1.61(dq,J=18.5,7.0Hz,6H),1.43(s,9H),1.26(s,60H),0.88(t,J=6.7Hz,6H).

[0233] Step 4: N 6 -((Azide-polyethylene glycol 2000-acetyl)-N 2 -(tert-Butyloxycarbonyl)-L-lysyl-L-glutamic acid di(octadecyl) ester (SA 2 Glu-BocLys-PEG 2000 -N 3 )

[0234]

[0235] Compound N 2 -(tert-Butyloxycarbonyl)-L-lysyl-L-glutamic acid dioctadecyl ester (33 mg, 0.0375 mmol) and NHS-PEG 2000 -N 3 (50 mg, 0.025 mmol) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), and 7 μL of triethylamine was added under stirring, and the reaction was carried out at room temperature for 12 h. TEA was removed by distillation under reduced pressure, and the DMSO was diluted and transferred to a dialysis bag with a molecular weight cutoff of 2000, and dialyzed with DMSO for 2 days, then dialyzed with pure water for 2 days, and freeze-dried to obtain a white powder product (68 mg, 94%). 1 HNMR (300 Hz, CDCl 3): δ7.14(d,J=6.3Hz,1H),6.93(d,J=7.9Hz,1H),5.23(d,J=7.9Hz,1H),4.57(td,J=8.0,4.9Hz,1H),4. 12(t,J=6.7Hz,2H),4.05(t,J=6.8Hz,3H),3.98(s,2H),3.88(s,1H),3.65(s,171H),3.44–3.36(m,3H) ,3.29(s,2H),2.47–2.33(m,3H),2.19(dt,J=13.6,6.9Hz,1H),2.05–1.93(m,1H),1.85(dt,J=13.2,6. 5Hz,1H),1.61(dq,J=15.7,7.5,7.1Hz,7H),1.44(s,9H),1.43(s,2H),1.26(s,62H),0.91–0.85(t,6H).

[0236] Step 5: N 6 -((Azide-polyethylene glycol 2000-acetyl)-L-lysyl-L-glutamic acid di(octadecanol) ester (SA 2 Glu-Lys-PEG 2000 -N 3 )

[0237]

[0238] Compound SA 2 Glu-BocLys-PEG 2000 -N 3 Dissolve in 0.6 mL of dichloromethane, add 0.2 mL of trifluoroacetic acid dropwise while stirring, and react at room temperature for 10 h. Remove the solvent under pressure, add 2 mL of saturated sodium bicarbonate and 1 mL of pure water, transfer to a dialysis bag with a molecular weight cutoff of 1000, dialyze with pure water for 2 days, and freeze-dry to obtain a white powder product (62 mg, 94%). 1 H NMR (300 Hz, CDCl 3): δ7.87(d,J=8.3Hz,1H),7.09(d,J=6.4Hz,1H),4.57(td,J=8.1,5.2Hz,1H),4.09(dt,J=19.2,6.8H z,4H),3.98(s,2H),3.88(dd,J=5.8,4.0Hz,1H),3.65(s,160H),3.40(q,J=4.6Hz,3H),3.30(q,J=7. 2,6.8Hz,2H),2.42-2.34(m,4H),2.18(dd,J=14.0,7.1Hz,1H),2.01(dt,J=14.2,7.0Hz,1H),1.94-1 .82(m,1H),1.59(dt,J=14.2,6.8Hz,7H),1.47(t,J=4.2Hz,2H),1.26(s,63H),0.88(t,J=6.5Hz,6H).

[0239] Step 6: N 6 -((Azide-polyethylene glycol 2000-acetyl)-N 2 -(Methyl-polyethylene glycol 1000-acetamido)-L-lysyl-L-glutamic acid di(octadecanol) ester (SA 2 Glu-(M-PEG 1000 )Lys-PEG 2000 -N 3 )

[0240]

[0241] Compound SA 2 Glu-Lys-PEG 2000 -N 3 (20 mg, 0.007 mmol), M-PEG 1000 -COOH (8 mg, 0.0077 mmol) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH, 4 mg, 0.014 mmol) were dissolved in 3 mL DMSO, and 20 μL N-methylimidazole (NMI) was added dropwise under stirring. The mixture was reacted at room temperature for 24 h. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3000, dialyzed against DMSO for 2 days and pure water for 2 days, and freeze-dried to obtain a white powder solid as the control compound SA 2 Glu-(M-PEG 1000 )Lys-PEG 2000 -N 3 (15 mg, yield: 74%). 1 H NMR (300 Hz, CDCl 3): δ8.13(s,1H),7.12(s,1H),6.90(d,J=7.7Hz,1H),5.55(d,J=7.8Hz,1H),4.59–4.51(m,1H),4.22(t,J=4.8Hz,2 H),4.12(dd,J=6.8,2.2Hz,2H),4.06(t,J=6.9Hz,3H),3.99(s,2H),3.82(s,2H),3.65(s,271H),3.56(d,J=5.1Hz, 3H),3.47(s,2H),3.38(s,5H),3.29(d,J=6.6Hz,2H),2.63(s,1H),2.48–2.28(m,6H),2.19(m,2H),2.01(dt,J=14 .7,7.3Hz,2H),1.87(s,1H),1.56(dd,J=14.7,7.2Hz,9H),1.45–1.38(m,3H),1.26(s,64H),0.88(t,J=6.7Hz,6H).

[0242] Embodiment 4: Synthesis

[0243] The specific operation steps refer to step 4 in Example 1, using the intermediate ZM-PEG 1000 -COOH replaces the raw material M-PEG 1000 -COOH, and the final yellow-brown powder is compound SA 2 -ZM. 1 H NMR (300 Hz, CDCl 3 ): δ7.57(s,1H),7.21(d,J=3.6Hz,1H),7.17(d,J=8.3Hz,2H),6.86(d,J=8.1Hz,2H),6.55(dd,J =3.4,1.8Hz,1H),4.46(d,J=13.5Hz,2H),4.14–4.00(m,7H),3.97(s,2H),3.88(s,2H),3.65(s, 290H),3.40(d,J=5.1Hz,4H),2.88(d,J=6.9Hz,2H),2.54–2.27(m,6H),2.18(dq,J=13.7,7.1,6 .6Hz,2H),2.06–1.87(m,2H),1.58(dd,J=13.4,6.7Hz,7H),1.25(s,65H),0.88(t,J=6.6Hz,6H).

[0244] Example 5: Synthesis of fluorescently modified multifunctional anchoring molecules

[0245] In order to facilitate the subsequent anchoring investigation of multifunctional anchoring molecules, DBCO-FITC was synthesized, and a fluorescently modified multifunctional anchoring molecule was synthesized through a click reaction.

[0246] 1. The synthesis method is as follows:

[0247] Step 1: N 6 -(Fluorenylmethoxycarbonyl)-N 2 Synthesis of -(tert-Butyloxycarbonyl)lysine methyl ester

[0248]

[0249] N 6- (Fluorenylmethoxycarbonyl)-N 2 -(tert-Butyloxycarbonyl)lysine (937 mg, 2.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 575 mg, 3.0 mmol) and 4-dimethylaminopyridine (DMAP, 48.8 mg, 0.4 mmol) were dissolved in a 10 mL methanol and tetrahydrofuran mixed solution (methanol:tetrahydrofuran = 1:1, v / v), and reacted at room temperature for 12 h. After the reaction was complete, the solvent was removed under reduced pressure, dissolved in 20 mL of dichloromethane, the organic phase was washed with appropriate amount of water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After purification by column chromatography (petroleum ether: ethyl acetate = 1:1), a white solid was obtained as the compound (880 mg, yield: 89%). 1 H NMR (300 Hz, CDCl 3 ): δ7.81(dd,J=7.5,1.4Hz,2H),7.74-7.68(m,2H),7.59(td,J=7.4,1.5Hz,2H),7 .40(td,J=7.5,1.5Hz,2H),5.80(d,J=10.8Hz,1H),5.66(t,J=7.3Hz,1H),4.45(d, J=7.0Hz,2H),4.27-4.18(m,2H),3.69(s,3H),3.17(dq,J=12.4,7.1Hz,1H),2.79( dq,J=12.5,7.1Hz,1H),1.77-1.58(m,2H),1.57-1.35(m,12H),1.33-1.24(m,1H).

[0250] Step 2: N 6 Synthesis of -(Fluorenylmethoxycarbonyl)lysine methyl ester

[0251]

[0252] N 6-(Fluorenylmethoxycarbonyl)-N 2 -(tert-Butoxycarbonyl)lysine methyl ester (880 mg, 1.825 mmol) was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added under stirring. The reaction was allowed to react at room temperature for 3 h. After the reaction was complete, the solvent was removed by distillation under reduced pressure. After dissolving in 20 mL of dichloromethane, the organic phase was washed with appropriate amounts of water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a white solid as the compound (640 mg, yield: 92%). 1 H NMR (300Hz, CDCl3): δ7.81 (dd, J=7.4, 1.5Hz, 2H), 7.70 (dd, J=7.4, 1.4Hz, 2H), 7.50 (td, J=7.5, 1.5Hz,2H),7.40(td,J=7.5,1.5Hz,2H),4.46(d,J=7.0Hz,2H),4.24(td,J=7.1,6.5,0.9Hz,1H), 3.80(dd,J=8.5,7.2Hz,1H),3.73–3.64(m,4H),3.56(dd,J=8.5,7.2Hz,1H),3.16(dq,J=12.3,7. 2Hz,1H),2.77(dq,J=12.3,7.1Hz,1H),1.81–1.66(m,2H),1.65-1.49(m,2H),1.48–1.32(m,2H).

[0253] Step 3: N 6 -(Fluorenylmethoxycarbonyl)-N 2 Synthesis of methyl lysine -(11,12-didehydro-γ-oxodibenzo[B,F]azocane-5(6H)-butyryl)

[0254]

[0255] N 6 -(Fluorenylmethoxycarbonyl)lysine methyl ester (211 mg, 0.55 mmol), DBCO-COOH (153 mg, 0.5 mmol), DMAP (7 mg, 0.06 mmol) were dissolved in 10 mL of dichloromethane, EDCI (144 mg, 0.75 mmol) was added under ice-water bath, and the mixture was reacted at room temperature for 12 h. After the reaction was complete, 10 mL of dichloromethane was added to dilute the mixture, the organic phase was washed with appropriate amount of water and saturated brine, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the compound (316 mg, yield: 94%) was obtained as a light blue solid after purification by column chromatography (dichloromethane: methanol = 100: 1). 1 H NMR (300 Hz, DMSO-d 6): δ8.19-8.07(m,1H),7.89(d,J=7.5Hz,2H),7.72-7.58(m,4H),7.55-7.21(m,11H),5. 16-4.94(m,1H),4.42-4.26(m,2H),4.21(d,J=6.9Hz,1H),4.15-3.99(m,1H),3.58(dd,J =19.9,11.8Hz,4H),3.05-2.82(m,2H),2.60(dt,J=15.6,7.9Hz,1H),2.42-2.21(m,1H) ,2.14-1.95(m,1H),1.87-1.68(m,1H),1.52(dd,J=15.6,7.9Hz,2H),1.41-1.10(m,5H).

[0256] Step 4: N 6 -((3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)aminothienyl)-N 2 Synthesis of -(11,12-didehydro-γ-oxodibenzo[B,F]azocane-5(6H)-butyryl)lysine methyl ester (DBCO-FITC)

[0257]

[0258] N 6 -(Fluorenylmethoxycarbonyl)-N 2 -(11,12-Didehydro-γ-oxodibenzo[B,F]azocane-5(6H)-butyryl)lysine methyl ester (270 mg, 0.4 mmol) was dissolved in 0.8 mL DMF, 0.2 mL piperidine was added dropwise under stirring, and the reaction was carried out at room temperature for 3 h. After the reaction is complete, 100 mL of ethyl acetate is added to dilute, washed with saturated brine (3×25 mL), dried over anhydrous sodium sulfate, and directly used in subsequent reactions after removing the solvent under reduced pressure. The crude product and FITC (189 mg, 0.48 mmol) are dissolved in a mixed solution of DCM / DMSO (4 mL / 0.8 mL), and 210 μL of DIPEA is added dropwise with stirring. The reaction is carried out at room temperature for 3 h. After the reaction is complete, 30 mL of DCM is added to dilute, washed with water (3×25 mL) and saturated brine (3×25 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (dichloromethane: methanol = 60:1) to obtain an orange-yellow solid, namely DBCO-FITC (150 mg, two-step yield: 45%). 1 HNMR (300 Hz, DMSO-d 6): δ10.15(s,2H),10.06(s,1H),8.31-8.10(m,2H),7.86-7.58(m,3H),7.49(td,J=6.6,2.8Hz,2H),7.43-7 .24(m,3H),7.17(d,J=8.2Hz,1H),6.68(d,J=2.1Hz,2H),6.64-6.52(m,4H),5.03(d,J=13.7Hz,1H),4.26-3 .86(m,2H),3.59(dd,J=14.3,10.6Hz,4H),3.35(s,3H),3.19-3.09(m,2H),2.71-2.54(m,1H),2.33(td,J=1 4.8,7.5Hz,1H),2.19-1.95(m,1H),1.80(dt,J=13.7,6.8Hz,1H),1.54(s,2H),1.26(td,J=6.8,3.2Hz,6H).

[0259] 2. Synthesis of Fluorescently Labeled Multifunctional Anchoring Molecules

[0260] DSPE-Lys(M-PEG 1000 )-PEG 2000 Synthesis of -FITC

[0261] DSPE-Lys(M-PEG 1000 )-PEG 2000 -N 3 (22 mg, 0.005366 mmol) and DBCO-FITC (6.7 mg, 0.008050 mmol) were dissolved in 3 mL of DCM / DMSO mixed solution (v:v=2:1) ​​and reacted at room temperature overnight. After the reaction was complete, DCM was removed under reduced pressure, 2 mL of DMSO was added for dilution, and then transferred to a dialysis bag with a molecular weight cutoff of 3000, dialyzed with DMSO for 2 days, dialyzed with pure water for 2 days, and freeze-dried to obtain DSPE-Lys (M-PEG 1000 )-PEG 2000 -FITC (13 mg, yield: 50%). 1 H NMR (300 MHz, CDCl 3)δ8.22(s,2H),7.56(s,4H),7.45(s,3H),7.37(s,3H),7.20(s,2H),7.14(s,3H),6.88-6.49(m,11H),5.99(s,1H),5.24(s,1H),5.10 (s,1H),4.46(s,5H),3.99(d,J=7.6Hz,5H),3.64(d,J=2.5Hz,252H),2.28(s,9H),1.43(s,3H),1.25(s,61H),0.87(t,J=6.6Hz,6H).

[0262] SA 2 Synthesis of Glu-Lys(M-PEG1000)-PEG2000-FITC

[0263] Detailed operation steps refer to DSPE-Lys(M-PEG 1000 )-PEG 2000 -FITC, freeze-dried to obtain SA 2 Glu-Lys(M-PEG 1000 )-PEG 2000 -FITC. 1 H NMR (400 MHz, CDCl 3)δ8.21(s,2H),8.06–7.92(m,2H),7.76(s,1H),7.66–7.50(m,4H),7.45(s,3H), 7.37(s,1H),7.23(s,3H),7.11(d,J=6.9Hz,2H),6.92(d,J=7.7Hz,2H),6.85(s,2 H),6.66(s,2H),5.97(dd,J=37.6,15.9Hz,3H),5.56(d,J=7.9Hz,1H),4.54(td,J =7.9,5.1Hz,4H),4.41(d,J=16.6Hz,3H),4.29(d,J=12.4Hz,2H),4.21(t,J=4.8H z,3H),4.12(qd,J=6.7,2.7Hz,4H),4.05(t,J=6.8Hz,3H),3.98(s,2H),3.81(s,2 H),3.78–3.49(m,269H),3.46(s,2H),3.38(s,3H),3.29(q,J=6.8Hz,2H),2.54–2 .29(m,8H),2.19(m,4H),2.01(dt,J=14.5,7.3Hz,3H),1.94–1.82(m,3H),1.61(t dd,J=7.3Hz,12H),1.45–1.37(m,4H),1.26(s,64H),0.90–0.86(t,J=6.6Hz,6H).

[0264] DSPE-Lys(ZM-PEG 1000 )-PEG 2000 Synthesis of -FITC

[0265] Detailed operation steps refer to DSPE-Lys(M-PEG 1000 )-PEG 2000 -FITC, freeze-dried to obtain DSPE-Lys(ZM-PEG 1000 )-PEG 2000 -FITC. 1 H NMR (300 MHz, CDCl 3)δ8.22(s,1H),7.70-7.52(m,3H),7.41(d,4H),7.19(s,2H),7.11(d,1H),6.99 (s,2H),6.82(s,3H),6.72(s,2H),6.62(s,4H),5.98(s,1H),5.32-5.00(m,1H) ,4.37(s,4H),4.14(s,3H),4.09-3.92(m,4H),3.63(d,254H),2.44(s,4H),2.2 9(q,6H),1.50-1.40(m,5H),1.25(s,65H),1.06(s,5H),0.86(t,J=6.6Hz,6H).

[0266] SA 2 Glu-Lys(ZM-PEG 1000 )-PEG 2000 Synthesis of -FITC

[0267] Detailed operation steps refer to DSPE-Lys(M-PEG 1000 )-PEG 2000 -FITC, freeze-dried to obtain SA 2 Glu-Lys(ZM-PEG 1000 )-PEG 2000 -FITC. 1 H NMR (300 MHz, CDCl 3 )δ7.56(s,4H),7.46(s,2H),7.34(s,2H),7.21(s,2H),7.14(s,3H),7.07(s,3H) ,6.85(s,4H),6.67(s,3H),6.55(s,1H),4.50(d,J=21.4Hz,7H),4.16-3.99(m,7H ),3.97(s,2H),3.64(s,265H),3.14(s,1H),2.38(q,J=7.0,6.4Hz,4H),2.14(s,7 H),1.98(s,7H),1.63(s,12H),1.43(s,4H),1.25(s,64H),0.88(t,J=6.6Hz,6H).

[0268] Example 6: Investigation of the safety of multifunctional anchoring molecules on cells

[0269] The safety of the multifunctional anchoring molecules in Examples 1 to 4 above on cells was determined by the CCK8 method. Taking T cells as an example, the specific operation was as follows:

[0270] The multifunctional anchoring molecule was dissolved in DMSO to prepare a 10 mM stock solution, and then diluted with 1640 RPMI medium to different concentrations of anchoring solution (2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM). T cells were dispersed in 1640 medium and cultured at 1×10 4 100 μL per well was plated on a 96-well plate and incubated in an incubator for 1-2 hours. After the incubation was complete, the plate was centrifuged and the supernatant was discarded. 100 μL of anchoring solution of different concentrations was added to each well and incubated in an incubator for 1 hour. After the incubation was complete, the plate was centrifuged and the supernatant was discarded. 200 μL of T cell complete medium was added to each well and incubated in an incubator for 12 hours. After the incubation was complete, 100 μL of 10% CCK8 solution mixed in advance was added to each well and incubated in an incubator for 1-3 hours. The absorbance at 450 nm was detected by an enzyme-labeled instrument. The calculation formula is, cell viability (%) = [A (anchor lipid)] - A (blank)] / [A (without anchor lipid) - A (blank)] x 100%, where A (anchor lipid) is the absorbance of the well with cells, CCK-8 solution and anchor protonated wells, A (blank) is the absorbance of the well with culture medium and CCK-8 solution but no cells, and A (without anchor lipid) is the absorbance of the well with cells, CCK-8 solution but no anchor lipid. The results are as follows Figure 2 As shown, each fluorescently labeled multifunctional anchoring molecule has no significant toxicity to T cells within 80 μM and has good cell safety.

[0271] Example 7: Preparation of cells anchored by multifunctional anchoring molecules

[0272] Taking T cells as an example, the multifunctional anchoring molecules DSPE-ZM or DSPE-m or SA in Examples 1 to 4 are 2 -ZM or SA 2 -m was mixed with freshly separated T precipitate, incubated at 37°C, washed with PBS and centrifuged to obtain cells anchored with multifunctional anchor molecules. Hoechst nuclear stain (10 mg / mL) was added and incubated at room temperature for 10 minutes. After washing and centrifugation, the cells were resuspended in PBS and spread in a confocal dish. The anchoring effect of membrane anchor molecules on cells was directly observed by laser confocal microscopy. The results are shown in Figure 3 As shown, DSPE-ZM, DSPE-m, SA 2 -ZM, SA 2 -m can be anchored on the surface of T cell membrane without significant endocytosis. In addition, DSPE-ZM and SA 2 -ZM compared to DSPE-m and SA without ligand ZM structure in the branched structure 2 -m has a stronger fluorescence intensity on the T cell membrane, indicating that the introduction of branched ligands can enhance the anchoring effect of anchor molecules on T cells.

[0273] Example 8: Investigation of the anchoring efficiency of multifunctional anchoring molecules

[0274] Taking T cells as an example, DSPE-ZM or DSPE-m or SA was obtained according to Example 7. 2 -ZM or SA 2 -m anchored T cells, and flow cytometry was used to examine the anchoring efficiency of the above anchoring molecules on T cells. Figure 4 As shown, DSPE-ZM and SA with ligand ZM introduced into the branched structure 2 -ZM compared to DSPE-m and SA without ligand ZM structure in the branched structure 2 -m has better anchoring efficiency to T cells, which indicates that the introduction of branched ligands can significantly improve the anchoring efficiency of anchoring molecules to T cells.

[0275] Example 9: Investigation of anchoring stability of multifunctional anchoring molecules

[0276] Taking T cells as an example, DSPE-ZM or DSPE-m or SA was obtained according to Example 7. 2 -ZM or SA 2 -m anchored T cells were cultured in culture medium for 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours. The MFI values ​​of T cells anchored by anchor molecules at different time points were detected by flow cytometry, and the anchoring stability of each anchor molecule was statistically calculated, where anchoring stability (%) = (MFI value of cells anchored by multifunctional anchor molecules / MFI value of cells anchored by multifunctional anchor molecules at 0 hours) × 100%. The results are shown in Figure 5 As shown, the anchoring molecule with the small molecule ligand ZM is more stable on the cell membrane than the anchoring molecule without the small molecule ligand, and has better anchoring stability.

[0277] Example 10: Multifunctional anchoring molecule anchoring enhances T cell activity

[0278] Taking T cells as an example, DSPE-ZM or DSPE-m anchored T cells were obtained according to Example 7, and cultured in a medium containing CD3 / CD28 antibodies and adenosine (ADO, 1 mM) for 24 h. The ELISA kit was used to detect the contents of IFN-γ, TNF-α, and GzmB in the cell supernatant. The results are shown in FIG. Figure 6 As shown, the anchor molecule with the small molecule ligand ZM can effectively relieve the immunosuppression of adenosine on T cells compared with the anchor molecule without the small molecule ligand.

[0279] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A compound represented by general formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts: in: R1 is selected from distearoylphosphatidylethanolamine (DSPE), 1,2-hexadecanoylphosphatidylethanolamine (DPPE), 1,2-tetradecanoylphosphatidylethanolamine (DMPE), 1,2-dioleoyl-SN-glycero-3-phosphatidylethanolamine (DOPE), cholesterol (Cholesterol), tocopherol (Tocopherol) and R2 is selected from H, D, halogen and C 1-30 alkyl; X is selected from azido, C 2-6 Alkynyl, Y is selected from a bond, O, S, carbonyl, C 1-6 Alkylene, imino, sulfone and sulfoxide groups, the C 1-6 Alkylene and imino are represented by one or more R Y replaced by; R Y are each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl; Z is selected from C 1-6 alkyl, The C 1-6 The alkyl group is replaced by one or more R Z replaced by; R Z are each independently selected from H, D, halogen, amino, hydroxyl and C 1-6 alkyl; n is any integer from 0 to 45; m is any integer from 25 to 120; p is 1, 2, 3, 4 or 5; q is 1, 2, 3 or 4.

2. The compound of formula I according to claim 1 or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: R2 is selected from H, D, halogen and C 9-21 alkyl; Preferably, R2 is selected from H, D, halogen and C 14-18 alkyl; More preferably, said R2 is selected from C 14-18 alkyl; Most preferably, said R2 is selected from tetradecyl, hexadecyl and octadecyl.

3. The compound of formula I according to claim 1 or 2, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: The q is 2.

4. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 3, characterized in that: The R1 is selected from distearoylphosphatidylethanolamine (DSPE), 1,2-hexadecanoylphosphatidylethanolamine (DPPE), 1,2-tetradecanoylphosphatidylethanolamine (DMPE), 5. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 4, characterized in that: The X is selected from azide, C 2-4 Alkynyl, Preferably, X is selected from azido, ethynyl, More preferably, said X is selected from an azide group.

6. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 5, characterized in that: The Y is selected from a bond, O, S, carbonyl, C 1-3 Alkylene, imino, sulfone and sulfoxide groups, the C 1-3 Alkylene and imino are represented by one or more R Y replaced by; Preferably, Y is selected from an imino group, wherein the imino group is replaced by one or more R Y replaced.

7. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 6, characterized in that: The R Y are each independently selected from H, D, halogen, amino, hydroxyl and C 1-3 alkyl; Preferably, the R Y are each independently selected from H.

8. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 7, characterized in that: The Z is selected from C 1-3 alkyl, The C 1-3 The alkyl group is replaced by one or more R Z replaced by; Preferably, Z is selected from methyl, The methyl group is replaced by one or more R Z replaced by; More preferably, Z is selected from 9. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 8, characterized in that: The R Z are each independently selected from H, D, halogen, amino, hydroxyl and C 1-3 alkyl; Preferably, the R Z are each independently selected from H.

10. The compound of formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt according to any one of claims 1 to 9, characterized in that: The n is any integer from 10 to 30, preferably any integer from 20 to 25, and more preferably 23.

11. The compound of formula I according to any one of claims 1 to 10, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: The m is any integer from 30 to 60, preferably any integer from 40 to 50, and more preferably 46.

12. The compound of formula I according to any one of claims 1 to 11, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: The p is 4.

13. A compound or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: The compound is selected from:

14. A cell, characterized in that The cell comprises the compound of any one of claims 1 to 13 or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt; Preferably, the cells are selected from primary cells or immortalized cells having a lipid membrane structure of humans or animals, preferably from tumor cells, neutrophils, T cells, mesenchymal stem cells, hematopoietic stem cells, natural killer cells, antigen presenting cells and macrophages, more preferably from T cells and neutrophils, and most preferably from T cells.

15. A cell medicine, characterized in that: The cell medicine comprises the cell and the bioactive molecule according to claim 14; Preferably, the bioactive molecule is selected from one or more of fluorescent probes, proteins and nanomedicines; Preferably, the fluorescent probe is selected from one or both of fluorescent dyes and AIE probes; Preferably, the fluorescent dye is selected from one or more of FITC, DIR and DIL; Preferably, the protein is selected from one or both of therapeutic antibodies and chemokines; Preferably, the therapeutic antibody is selected from one or both of PD-1 monoclonal antibody and PD-L1 monoclonal antibody; Preferably, the chemokine is selected from CXCL-8; Preferably, the nanomedicine is selected from nanoparticles loaded with a therapeutic agent; Preferably, the nanoparticles are liposomes, nanovesicles, solid lipid nanoparticles or micelles with a particle size of 1 nm to 1000 nm; Preferably, the therapeutic agent is selected from one or more of avasimibe, paclitaxel and PD-1 monoclonal antibody.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of a compound according to any one of claims 1 to 13, or a stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt thereof, or a therapeutically effective amount of a cell according to claim 14 or a therapeutically effective amount of a cell drug according to claim 15.

17. A compound according to any one of claims 1 to 13, or a stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt thereof, or a cell according to claim 14, or a cell drug according to claim 15, or a pharmaceutical composition according to claim 16, for use in preventing and / or treating a disease; Preferably, the disease is selected from tumors and autoimmune diseases; Preferably, the tumor includes melanoma, glioma, lung cancer, breast cancer and ovarian cancer; Preferably, the autoimmune diseases include stroke, arthritis and systemic lupus erythematosus.

18. Use of the compound according to any one of claims 1 to 13, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the cell according to claim 14, or the cell drug according to claim 15, or the pharmaceutical composition according to claim 16 for preventing and / or treating diseases; Preferably, the disease is selected from tumors and autoimmune diseases; Preferably, the tumor includes melanoma, glioma, lung cancer, breast cancer and ovarian cancer; Preferably, the autoimmune diseases include stroke, arthritis and systemic lupus erythematosus.

19. Use of the compound according to any one of claims 1 to 13, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the cell according to claim 14, or the cell drug according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a drug for preventing and / or treating a disease; Preferably, the disease is selected from tumors and autoimmune diseases; Preferably, the tumor includes melanoma, glioma, lung cancer, breast cancer and ovarian cancer; Preferably, the autoimmune diseases include stroke, arthritis and systemic lupus erythematosus.

20. Use of the compound according to any one of claims 1 to 13, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the cell according to claim 14, or the cell drug according to claim 15, or the pharmaceutical composition according to claim 16 in in vivo visualization of cells.

21. Use of the compound according to any one of claims 1 to 13, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or the cell according to claim 14, or the cell drug according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of an agent for in vivo visualization of cells.

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