A dual-targeting molecule based on hyaluronic acid and glucose and its preparation method and application

Abstract

The present invention discloses a dual-targeting molecule based on hyaluronic acid and glucose, a preparation method and application thereof, in particular, a dual-targeting fluorescent probe and anti-tumor drug based on hyaluronic acid and glucose. The dual-targeting fluorescent probe utilizes the interaction between HA-CD44 and Glc-GLUT1 to simultaneously target the CD44 and GLUT1 receptors of tumors (e.g., lung tumors), and can deliver fluorescent molecules into tumor cells in a targeted manner, reducing the aggregation of fluorescent molecules in normal cells; at the same time, the tumor cell endocytosis efficiency of fluorescent molecules can be improved. The chemical structures of the two targeting molecules HA and glucose in the dual-targeting fluorescent probe are clear, and the covalent bond connection sites and compositions are clear. This feature provides dual-targeting molecules with very good research and application value in drug delivery system applications and innovative drug development.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine technology, and in particular to a dual-targeting molecule based on hyaluronic acid and glucose, a preparation method and application thereof, and in particular to a dual-targeting fluorescent probe and anti-tumor drug based on hyaluronic acid and glucose. Background Art

[0002] Currently, the main means of tumor treatment include chemotherapy, radiotherapy, etc. Although the research on tumor treatment methods and therapeutic drugs has made great progress, it is still difficult to overcome this major disease that threatens human health. Traditional chemotherapy, as the main means of tumor treatment, has extremely strong toxic side effects. Therefore, developing a drug delivery system with tumor targeting capabilities to improve the effective targeted enrichment of chemotherapy drugs in lesions has become a problem that needs to be urgently solved in clinical practice.

[0003] Sugar molecules are the most abundant class of biomolecules on earth. Glycoconjugates exist on the cell surface in free form or in the form of glycoproteins, glycolipids, etc. The interaction between sugar molecules and receptors plays a vital role in many physiological processes such as fertilization, immune defense, cell differentiation, cell-matrix interaction and cell-cell adhesion. Lectins, as one of the receptors for sugar molecules, are proteins of non-immune origin that bind to specific carbohydrates. Animal lectins are involved in the transport and clearance of glycoproteins, development, immune defense, fertility and other biological processes. In terms of sensitivity and selectivity, sugar molecules have shown great potential in mediating cell surface interactions. As detection tools, sugar chains and glycoconjugates have been widely used in biological imaging applications, early tumor detection imaging, lectin detection or interaction, bacterial detection, sugar interactions, etc.

[0004] Hyaluronan (HA) is a linear polysaccharide composed of repeated disaccharide units of β-1,4-linked N-acetylglucosamine (β-D-GlcNAc) and β-1,3-linked glucuronic acid (β-D-GlcA), with no sulfation modification and no covalently linked core protein. The CD44 receptor belongs to the transmembrane glycoprotein family and has great potential for active targeted therapy. At the same time, CD44 is the main receptor for HA. CD44 is expressed at low levels on the surface of healthy cells (such as epithelial cells, hematopoietic cells, and neural cells). In contrast, CD44 variants are overexpressed on the surface of various tumor cells, including breast tumors, squamous tumors, ovarian tumors, and colon tumors. HA participates in the occurrence and development of tumors, inflammatory responses, and regulation of intracellular signaling pathways by interacting with CD44 receptors on the surface of tumor cells.

[0005] Human glucose transporter 1 (GLUT1) is significantly overexpressed in many human tumors, including liver tumors, pancreatic tumors, breast tumors, esophageal tumors, brain tumors, kidney tumors, lung tumors, skin tumors, colorectal tumors, endometrial tumors, ovarian tumors, and cervical tumors. GLUT1 has been developed as an effective clinical drug delivery target. Through GLUT1-mediated endocytosis, drug delivery nanosystems targeting GLUT1 can effectively enter target cells. Glucose (Glc), as a substrate of GLUT1, has the advantages of small molecules, good biocompatibility, and non-toxicity, and can also be used as a targeting ligand for tumor-targeted drugs.

[0006] The targeting ability of the nano-drug delivery system brought by a single targeting ligand is often limited, so the introduction of multi-targeting ligands can enable drugs to enter cells through endocytosis mediated by different receptor ligands, improving the drug's internalization ability to a certain extent. Many tumor surfaces have multiple carbohydrate receptors at the same time. For example, breast tumor cells have CD44 and GLUT1 receptors at the same time. By using the interaction between HA-CD44 and Glc-GLUT1, HA-glucose dual-targeting molecules are designed to be coupled with the fluorescent molecule rhodamine, which can be used for tumor cell tracing of dual-targeting molecules. At present, most of the breast tumor dual-targeting systems related to HA use HA and folic acid as dual-targeting molecules. There are no related reports on the dual-targeting molecular drug delivery system using HA and glucose that interact with HA-CD44 and Glc-GLUT1. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a dual-targeting molecule based on hyaluronic acid and glucose, and a preparation method and application thereof.

[0008] In a first aspect of the present invention, a method for preparing a dual-targeting molecule based on hyaluronic acid and glucose (e.g., the dual-targeting fluorescent probe and the dual-targeting anti-tumor compound of the present invention) is provided, which comprises the following steps: preparing an intermediate M1 from hyaluronic acid or its oligosaccharides (especially hyaluronic acid oligosaccharides), preparing an intermediate M2 from glucose, preparing an intermediate M3 from a target (e.g., a target compound, a peptide, a protein), and then performing a click reaction between M1, M2 and M3 to obtain a target product;

[0009]

[0010] in,

[0011] n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0012] a, b, c, d, e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0013] M is H or an alkali metal (e.g., Na, K);

[0014] L' is an optional suitable group (eg -CH2NH-);

[0015] T is the target residue (eg, a fluorophore residue (F), an anti-tumor drug residue (D)).

[0016] In one embodiment of the present invention, the target compound is a fluorophore, for example, an inorganic fluorescent material or an organic small molecule fluorescent dye. Among them, the inorganic fluorescent materials mainly include rare earth fluorescent materials, including representative lanthanide luminescent elements-europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd), etc., and luminescent quantum dots such as CdSe, CdTe, etc. Organic small molecule fluorescent materials such as coumarins, fluoresceins, rhodamines, phenanthroline, naphthylimide, fluorine boron fluorescent, cyanine dyes, etc.

[0017] In some embodiments of the present invention, the fluorophore is an organic small molecule fluorescent dye, such as a rhodamine dye.

[0018] In some embodiments of the invention, the fluorophore residue has the following structure:

[0019]

[0020] Wherein, R1 to R6 are independently selected from: H, C1-6 alkyl (eg, methyl, ethyl).

[0021] In another embodiment of the present invention, the target is an anti-tumor drug, such as cytotoxic antibiotics, plant alkaloids, antimetabolites, alkylating agents, platinum compounds, protein kinase inhibitors, immune checkpoint regulators, etc.

[0022] Specifically, the cytotoxic antibiotics include but are not limited to: ixabepilone, mitomycin, plicamycin, bleomycin, pyrantelone, amrubicin, valrubicin, pirarubicin, mitoxantrone, idarubicin, daunorubicin, aclarubicin, epirubicin, daunorubicin, doxorubicin and dactinomycin, etc.

[0023] Specifically, the plant alkaloids include but are not limited to: trabectedin, cabazitaxel, paclitaxel polyaniline, docetaxel, paclitaxel, demeclocycline, teniposide, etoposide, vinforin, vinflunine, vinorelbine, vindesine, vincristine and vinblastine.

[0024] Specifically, the antimetabolites include but are not limited to: floxuridine, trifluridine, tegafur, fluorouracil, decitabine, azacitidine, capecitabine, gemcitabine, carmofur, cytarabine, nelarabine, clofarabine, fludarabine, cladribine, thioguanine, mercaptopurine, pralatrexate, pemetrexed, raltitrexed and methotrexate, etc.

[0025] Specifically, the alkylating agent includes but is not limited to: dacarbazine, temozolomide, pipobromide, mitropromol, etoside, uracil mustard, ranimustine, nimustine, flutemustine, streptozotocin, semustine, lomustine, carmustine, carboquinone, triazinequinone, thiotepa, mannosulfuron, trothiosulfuron, busulfan, bendamustine, prednimustine, trofosfamide, ifosfamide, methylchlorambucil, melphalan, chlorambucil and cyclophosphamide, etc.

[0026] Specifically, the platinum compound includes but is not limited to: cisplatin, carboplatin, oxaliplatin, satraplatin and polyplatin.

[0027] Specifically, the protein kinase inhibitors include but are not limited to: BTK inhibitors, PI3K inhibitors, SYK inhibitors, JAK inhibitors and the like.

[0028] Specifically, the immune checkpoint regulators include but are not limited to: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, OX-40 agonists, CD137 agonists, LAG-3 inhibitors, and IDO inhibitors.

[0029] In some embodiments, the PD-1 inhibitor is selected from nivolumab, pidilizumab, pembrolizumab, AMP-224 (CAS#1422184-00-6), STI-A1110, AMP-514 (MEDI0680, CAS#1642374-69-3), TSR-043 and AUNP-12 (AUR-012, Aurigene-012, Aurigene NP-12).

[0030] In some embodiments, the PD-L1 inhibitor is selected from RG 7446, BMS-936559 (MDX 1105, CAS# 1422185-22-5), MSB0010718C, STI-A1010, avelumab, atezolizumab, and durvalumab.

[0031] In some embodiments, the CTLA-4 inhibitor is selected from ipilimumab and tremelimumab.

[0032] In some embodiments, the OX40 agonist is selected from anti-OX40, TIM3 antibodies, and Immutune IMP701.

[0033] In some embodiments, the CD137 agonist is selected from urelumab and utomilumab.

[0034] In some embodiments, the LAG-3 inhibitor is BMS-986016 (CAS #1683572-29-3).

[0035] In some embodiments, the IDO inhibitor is selected from GDC-0919 (CAS#1402836-58-1), indoximod, 1-methyl-D-tryptophan (NSC-721782), NLG919 (CAS#1402836-58-1) epacadostat, and norharman.

[0036] In some embodiments of the present invention, the target compound is a fluorophore, for example, M3 has the following structure:

[0037]

[0038] Wherein, R1 to R6 are independently selected from: H, C1-6 alkyl (eg, methyl, ethyl).

[0039] Specifically, n is an integer of 3-10, particularly 4-6, and preferably 5.

[0040] Specifically, a is an integer of 1-5, particularly 2-4, and preferably 3.

[0041] Specifically, b is an integer of 1-5, particularly 2-4, and preferably 3.

[0042] Specifically, c is an integer of 1-5, particularly 2-4, and preferably 3.

[0043] Specifically, d is an integer of 1-5, particularly 2-4, and preferably 3.

[0044] Specifically, e is an integer of 1-5, particularly 1-3, and preferably 2.

[0045] In one embodiment of the present invention, R3 and R4 are both H.

[0046] In another embodiment of the present invention, R3 and R4 are both methyl.

[0047] In one embodiment of the present invention, R1 and R5 are both H, and R2 and R6 are both methyl or ethyl.

[0048] In another embodiment of the present invention, R1 to R4 are all methyl groups.

[0049] In another embodiment of the present invention, R1 to R4 are all ethyl groups.

[0050] Specifically, the click reaction is carried out in the presence of a copper catalyst (e.g., (CH3CN)4CuBF4, CuSO4, CuBr, CuI); the click reaction system also includes a reducing agent, such as ascorbic acid; in some embodiments of the present invention, the click reaction is carried out in the presence of copper sulfate and ascorbic acid.

[0051] Specifically, the click reaction temperature is 20-30°C, such as room temperature.

[0052] Specifically, the click reaction time is 24-60 hours, for example 24, 36, 42, 48, 54, 60 hours.

[0053] In some embodiments of the present invention, the preparation method comprises the following reaction route:

[0054]

[0055] The reactions in the above steps (A1) and (A2) are click reactions, and the conditions are as described above.

[0056] Specifically, the above step (A1) comprises: dissolving compounds M3 and M2 in solvent A1, adding copper sulfate and ascorbic acid solution, and reacting.

[0057] Specifically, the solvent A1 can be a mixture of dichloromethane, water, and methanol (for example, in a ratio of 3:1:3, v / v).

[0058] Specifically, the reaction temperature in the above step (A1) is 20-30°C, such as room temperature.

[0059] Specifically, the reaction time in the above step (A1) is 24-60 hours, for example 24, 36, 42, 48, 54, 60 hours, especially 24 hours.

[0060] Specifically, step (A2) comprises: dissolving compounds M4 and M1 in solvent A2, adding copper sulfate and ascorbic acid solution, and reacting.

[0061] Specifically, the solvent A2 may be a mixture of water and methanol (eg, in a ratio of 1:1, v / v).

[0062] Specifically, the reaction temperature in the above step (A2) is 20-30 ° C, such as room temperature.

[0063] Specifically, the reaction time in the above step (A2) is 24-60 hours, for example 24, 36, 42, 48, 54, 60 hours, especially 48 hours.

[0064] Specifically, the preparation of intermediate M1 includes the following reaction route:

[0065]

[0066] Wherein, R7 is C1-6 alkyl (such as methyl);

[0067] R8 is a hydroxyl protecting group (e.g., acetyl);

[0068] →Indicates that step (B6) is optional (present when n>1) (may be present or absent, and may be carried out in one step or in multiple steps).

[0069] Specifically, the above step (B1) comprises: compound 6 reacts with alcohol and hydroxyl protecting agent respectively to obtain compound 7.

[0070] Specifically, the alcohol may be a C1-3 alcohol, such as methanol or ethanol.

[0071] Specifically, the hydroxyl protecting agent can be appropriately selected according to R8. For example, when R8 is acetyl, the hydroxyl protecting agent can be acetyl chloride or acetic anhydride.

[0072] Specifically, the above step (B2) comprises: (in solvent B1) compound 7 is mixed with DMAPA, and reacted under the protection of an inert gas to obtain compound 8.

[0073] Specifically, the solvent B1 is THF.

[0074] Specifically, the inert gas is argon.

[0075] Specifically, the reaction temperature in the above step (B2) is 20-30°C, such as room temperature.

[0076] Specifically, the reaction time in the above step (B2) is 0.5-6 hours, for example 1 hour.

[0077] Specifically, the above step (B3) comprises: (in solvent B2) compound 8 is mixed with trichloroacetonitrile and a base, and reacted under the protection of an inert gas to obtain compound 9.

[0078] Specifically, the solvent B2 is dichloromethane.

[0079] Specifically, the base is a strong organic base, such as 1,8-diazabicycloundec-7-ene (DBU).

[0080] Specifically, the inert gas is argon.

[0081] Specifically, the reaction temperature in the above step (B3) is 20-30°C, such as room temperature.

[0082] Specifically, the reaction time in the above step (B3) is 0.5-6 hours, for example 2 hours.

[0083] Specifically, the above step (B4) comprises: (in solvent B3) reacting compound 9 and compound The mixture is mixed and reacted in the presence of an acid under the protection of an inert gas to obtain compound 11.

[0084] Specifically, the solvent B3 is dichloromethane.

[0085] Specifically, the acid is a strong organic acid, such as trifluoromethanesulfonic acid.

[0086] Specifically, the inert gas is argon.

[0087] Specifically, the reaction temperature in the above step (B4) is 20-30°C, such as room temperature.

[0088] Specifically, the reaction time in the above step (B4) is 6-18 hours, for example 12 hours.

[0089] Specifically, the above step (B5) can adopt any suitable deprotection step, for example, compound 11 is mixed with hydrogen peroxide solution and LiOH, reacted at 0°C and then at room temperature, methanol and NaOH solution are added, and reacted at room temperature to obtain compound 12.

[0090] The above step (B6) may or may not exist, and may be carried out in one step or in multiple steps. For example, when n=1, step (B6) does not exist; when n=2, the reaction route of step (B6) is:

[0091]

[0092] That is, step (B6) is carried out in one step; when n=3, the reaction route of step (B6) is:

[0093]

[0094] That is, step (B6) is performed in multiple steps; and by analogy, M1 with different n values ​​is obtained by gradually extending the GlcNAc-GlcA disaccharide repeating unit at the non-reducing end of the sugar.

[0095] If necessary (n>1), the above step (B6) includes: using compound 12 as the starting substrate, reacting by (stepwise) adding sugar nucleotides and GlcA-transferase (GA-T) and GlcNAc-transferase (GN-T) to prepare the desired M1.

[0096] In one embodiment of the present invention, the GN-T is a hyaluronan synthase (Hyaluronan Synthase from Pasteurella multocoida) mutant having only GlcNAc-transferase catalytic activity.

[0097] In one embodiment of the present invention, the GA-T is a hyaluronan synthase (Hyaluronan Synthase from Pasteurella multocoida) mutant having only GlcA-transferase catalytic activity.

[0098] Specifically, the enzymatic reaction system includes: MgCl2, buffer (such as Tris-HCl), and GN-T / GA-T.

[0099] Specifically, the reaction temperature in step (B6) is 25-35°C, such as 25, 38, 30, 32, 35°C, especially 30°C.

[0100] Specifically, the reaction time in step (B6) is 6-24 hours, for example 12 hours.

[0101] Specifically, the preparation of intermediate M2 includes the following reaction route:

[0102]

[0103] Wherein, R9 is a hydroxyl protecting group (eg, acetyl).

[0104] Specifically, the above step (C1) includes: compound 13 (glucose) reacts with a hydroxyl protecting agent to obtain compound 14.

[0105] Specifically, the hydroxyl protecting agent can be appropriately selected according to R9. For example, when R9 is an acetyl group, the hydroxyl protecting agent can be acetyl chloride or acetic anhydride.

[0106] Specifically, the reaction temperature in the above step (C1) is 110-140°C, for example 110, 120, 130, 140°C.

[0107] Specifically, the reaction time in the above step (C1) is 1-6 hours, for example 2 hours.

[0108] Specifically, the above step (C2) comprises: (in solvent C1) compound 14 is mixed with ammonium acetate, and reacted to obtain compound 15.

[0109] Specifically, the solvent C1 is a mixture of methanol and tetrahydrofuran (for example, in a ratio of 1:1, v / v).

[0110] Specifically, the reaction temperature in the above step (C2) is 20-30°C, such as room temperature.

[0111] Specifically, the reaction time in the above step (C2) is 6-24 hours, for example 12 hours.

[0112] Specifically, the above step (C3) comprises: (in solvent C2) compound 15 is mixed with trichloroacetonitrile and a base, and reacted to obtain compound 16.

[0113] Specifically, the solvent C2 is dichloromethane.

[0114] Specifically, the base is a strong organic base, such as 1,8-diazabicycloundec-7-ene (DBU).

[0115] Specifically, the reaction temperature in the above step (C3) is 20-30°C, such as room temperature.

[0116] Specifically, the reaction time in the above step (C3) is 1-8 hours, for example 4 hours.

[0117] Specifically, the above step (C4) comprises: (in solvent C3) compound 16 and compound The mixture is mixed and reacted in the presence of an acid under the protection of an inert gas to obtain compound 17.

[0118] Specifically, the solvent C3 is dichloromethane.

[0119] Specifically, the acid is a strong organic acid, such as trifluoromethanesulfonic acid.

[0120] Specifically, the inert gas is argon.

[0121] Specifically, the reaction in the above step (C4) is carried out under ice bath conditions.

[0122] Specifically, the reaction time in the above step (C4) is 6-24 hours, for example 12 hours.

[0123] Specifically, the above step (C5) can adopt any suitable deprotection step, for example, mixing compound 16 with an alcohol (such as methanol), adjusting the pH to alkaline (such as pH 9) (such as by sodium methoxide), and reacting.

[0124] Specifically, the preparation of intermediate M3 includes the following reaction route:

[0125]

[0126] Among them, R 10 is an amino protecting group (such as a Boc group).

[0127] Specifically, the above step (D1) comprises: (in solvent D1) mixing compounds 21 and 22, adding DIPEA, EDCI and DMAP, and reacting to obtain compound 23.

[0128] Specifically, the solvent D1 is dichloromethane.

[0129] Specifically, the reaction temperature in the above step (D1) is 20-30°C, such as room temperature.

[0130] Specifically, the reaction time in the above step (D1) is 6-24 hours, for example 12 hours.

[0131] Specifically, the above step (D2) may adopt any suitable deprotection step, for example, compound 23 is reacted with an acid (eg, trifluoroacetic acid).

[0132] Specifically, the above step (D3) comprises: (in solvent D2) mixing compound 23 and compound 5, adding DIPEA, EDCI and DMAP, and reacting to obtain compound M3.

[0133] Specifically, the solvent D2 is dichloromethane.

[0134] Specifically, the reaction temperature in the above step (D3) is 20-30°C, such as room temperature.

[0135] Specifically, the reaction time in the above step (D3) is 6-24 hours, for example 12 hours.

[0136] Specifically, the preparation of compound 5 includes the following reaction route:

[0137]

[0138] Among them, R 11 is a hydroxyl protecting group (such as p-toluenesulfonyl);

[0139] R 12 It is C1-6 alkyl (such as methyl).

[0140] Specifically, the above step (E1) includes: in solvent E1, mixing compound 1 with propargyl bromide and a base, reacting to obtain compound 2; more specifically, step (1) includes: in solvent E1, mixing compound 1 with a base to obtain component 1; dissolving propargyl bromide in solvent E1 to obtain component 2; adding component 2 to component 1, reacting to obtain compound 2.

[0141] Specifically, the solvent E1 is tetrahydrofuran.

[0142] Specifically, the base is a strong organic base, such as potassium tert-butoxide.

[0143] Specifically, the above step (E2) comprises: compound 2 reacts with a hydroxyl protecting agent to obtain compound 3.

[0144] Specifically, the hydroxyl protecting agent can be based on R 11 Make appropriate selections, for example, R 11 When it is p-toluenesulfonyl, the hydroxyl protecting agent may be p-toluenesulfonyl chloride.

[0145] Specifically, the above step (E3) comprises: (in solvent E2) compound 3 and compound The base is mixed and reacted to obtain compound 4.

[0146] Specifically, the solvent E2 is N,N-dimethylformamide.

[0147] Specifically, the base is an organic weak base, such as potassium carbonate.

[0148] Specifically, the reaction temperature in the above step (E3) is 60-100°C, for example 60, 70, 75, 80, 85, 90, 100°C.

[0149] Specifically, the reaction time in the above step (E3) is 12-36 hours, for example 18 hours.

[0150] Specifically, the above step (E4) comprises: mixing compound 4 with alcohol, adding a base, and reacting to obtain compound 5.

[0151] Specifically, the alcohol may be a C1-3 alcohol, such as methanol or ethanol.

[0152] Specifically, the base is a strong organic base, such as potassium hydroxide.

[0153] Specifically, the reaction temperature in the above step (E4) is 60-100°C, for example 60, 70, 75, 80, 85, 90, 100°C.

[0154] Specifically, the reaction time in the above step (E4) is 1-6 hours, for example 2 hours.

[0155] In the second aspect of the present invention, intermediate compounds M1, M3, and M4 are provided, which can be used to prepare dual-targeted fluorescent probes or used in the method described in the first aspect, and have the following structure:

[0156]

[0157] in,

[0158] n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0159] a, b, c, d, e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0160] M is H or an alkali metal (e.g., Na, K);

[0161] R1 to R6 are independently selected from: H, C1-6 alkyl (eg methyl, ethyl).

[0162] Specifically, n is an integer of 3-10, particularly 4-6, and preferably 5.

[0163] Specifically, a is an integer of 1-5, particularly 2-4, and preferably 3.

[0164] Specifically, b is an integer of 1-5, particularly 2-4, and preferably 3.

[0165] Specifically, c is an integer of 1-5, particularly 2-4, and preferably 3.

[0166] Specifically, d is an integer of 1-5, particularly 2-4, and preferably 3.

[0167] Specifically, e is an integer of 1-5, particularly 1-3, and preferably 2.

[0168] In one embodiment of the present invention, R3 and R4 are both H.

[0169] In another embodiment of the present invention, R3 and R4 are both methyl.

[0170] In one embodiment of the present invention, R1 and R5 are both H, and R2 and R6 are both methyl or ethyl.

[0171] In another embodiment of the present invention, R1 to R4 are all methyl groups.

[0172] In another embodiment of the present invention, R1 to R4 are all ethyl groups.

[0173] In some embodiments of the present invention, intermediate M1 has the following structure:

[0174]

[0175] In some embodiments of the present invention, intermediate M3 has the following structure:

[0176]

[0177] In some embodiments of the present invention, intermediate M4 has the following structure:

[0178]

[0179] In the third aspect of the present invention, a method for preparing intermediate compounds M1, M3, and M4 is provided, as described in the first aspect of the present invention.

[0180] In the fourth aspect of the present invention, a dual-targeting molecule is provided, which includes a recognition group, a linker and a target residue, wherein the recognition group and the target residue are covalently linked via the linker, the recognition group includes hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue) and a glucose residue, and the target residue can be a reporter group or a drug residue.

[0181] Specifically, the dual targeting molecule has the following structure:

[0182]

[0183] in,

[0184] HA is hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue);

[0185] Glc is a glucose residue;

[0186] T is the target residue;

[0187] L is a linking group.

[0188] Specifically, hyaluronic acid and its oligosaccharide residues (especially hyaluronic acid oligosaccharide residues), glucose residues, and target substance residues have the definitions described in the first aspect of the present invention.

[0189] In some embodiments of the present invention, the hyaluronic acid oligosaccharide residue has the following structure:

[0190]

[0191] Wherein, n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0192] M is H or an alkali metal (eg, Na, K).

[0193] Specifically, n is an integer of 3-10, particularly 4-6, and preferably 5.

[0194] In some embodiments of the present invention, the glucose residue is

[0195] In some embodiments of the present invention, the linking group has the following structure:

[0196]

[0197] wherein a, b, c, d, and e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0198] Specifically, a is an integer of 1-5, particularly 2-4, and preferably 3.

[0199] Specifically, b is an integer of 1-5, particularly 2-4, and preferably 3.

[0200] Specifically, c is an integer of 1-5, particularly 2-4, and preferably 3.

[0201] Specifically, d is an integer of 1-5, particularly 2-4, and preferably 3.

[0202] Specifically, e is an integer of 1-5, particularly 1-3, and preferably 2.

[0203] In some embodiments of the present invention, the dual targeting molecule has the following structure:

[0204]

[0205] in,

[0206] n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0207] a, b, c, d, e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0208] M is H or an alkali metal (e.g., Na, K);

[0209] L' is an optional suitable group (eg, -CH2NH-).

[0210] In some embodiments of the present invention, T is a fluorophore residue, and the dual-targeting molecule is a dual-targeting fluorescent probe, as described in the fifth aspect of the present invention; in other embodiments of the present invention, T is an anti-tumor drug residue, and the dual-targeting molecule is a dual-targeting drug molecule, as described in the eighth aspect of the present invention.

[0211] In the fifth aspect of the present invention, a dual-targeting fluorescent probe is provided, which includes a recognition group, a reporter group and a linker, wherein the recognition group and the reporter group are covalently linked via the linker, the recognition group includes hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue) and a glucose residue, and the reporter group is a fluorophore residue.

[0212] Specifically, the dual-targeting fluorescent probe has the following structure:

[0213]

[0214] in,

[0215] HA is hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue);

[0216] Glc is a glucose residue;

[0217] F is the fluorophore residue;

[0218] L is a linking group.

[0219] In some embodiments of the present invention, the hyaluronic acid oligosaccharide residue has the following structure:

[0220]

[0221] Wherein, n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0222] M is H or an alkali metal (eg, Na, K).

[0223] Specifically, n is an integer of 3-10, particularly 4-6, and preferably 5.

[0224] In some embodiments of the present invention, the glucose residue is

[0225] Specifically, the fluorophore as a signal generating part can be an inorganic fluorescent material or an organic small molecule fluorescent dye. Among them, inorganic fluorescent materials mainly include rare earth fluorescent materials, including representative lanthanide luminescent elements-europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd), etc., and luminescent quantum dots such as CdSe, CdTe, etc. Organic small molecule fluorescent materials such as coumarins, fluoresceins, rhodamines, phenanthroline, naphthylimide, fluorine boron fluorescent, cyanine dyes, etc.

[0226] In some embodiments of the present invention, the fluorophore is an organic small molecule fluorescent dye, such as a rhodamine dye.

[0227] In some embodiments of the invention, the fluorophore residue has the following structure:

[0228]

[0229] Wherein, R1 to R6 are independently selected from: H, C1-6 alkyl (eg, methyl, ethyl).

[0230] In one embodiment of the present invention, R3 and R4 are both H.

[0231] In another embodiment of the present invention, R3 and R4 are both methyl.

[0232] In one embodiment of the present invention, R1 and R5 are both H, and R2 and R6 are both methyl or ethyl.

[0233] In another embodiment of the present invention, R1 to R4 are all methyl groups.

[0234] In another embodiment of the present invention, R1 to R4 are all ethyl groups.

[0235] In one embodiment of the present invention, the fluorophore residue is

[0236] In some embodiments of the present invention, the linking group has the following structure:

[0237]

[0238] wherein a, b, c, d, and e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0239] Specifically, a is an integer of 1-5, particularly 2-4, and preferably 3.

[0240] Specifically, b is an integer of 1-5, particularly 2-4, and preferably 3.

[0241] Specifically, c is an integer of 1-5, particularly 2-4, and preferably 3.

[0242] Specifically, d is an integer of 1-5, particularly 2-4, and preferably 3.

[0243] Specifically, e is an integer of 1-5, particularly 1-3, and preferably 2.

[0244] Specifically, the fluorescent probe has the following structure:

[0245]

[0246] in,

[0247] n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0248] a, b, c, d, e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0249] M is H or an alkali metal (e.g., Na, K);

[0250] L' is an optional suitable group (eg -CH2NH-);

[0251] F is a fluorophore residue.

[0252] In some embodiments of the present invention, the fluorescent probe has the following structure:

[0253]

[0254] In one embodiment of the present invention, the fluorescent probe has the following structure:

[0255]

[0256] In the sixth aspect of the present invention, there is provided the use of the dual-targeting fluorescent probe described in the fifth aspect in the preparation of tumor diagnostic agents and tumor tracers, which can be used for targeted fluorescence imaging of tumors, surgical navigation, tumor metastasis monitoring, etc.

[0257] Specifically, the tumors include, but are not limited to, liver tumors, pancreatic tumors, breast tumors, esophageal tumors, brain tumors, kidney tumors, lung tumors (e.g., non-small cell lung tumors), skin tumors, colon tumors, colorectal tumors, endometrial tumors, ovarian tumors, cervical tumors, etc.

[0258] In some embodiments of the invention, the tumor is a lung tumor (eg, a non-small cell lung tumor).

[0259] In the seventh aspect of the present invention, a detection reagent is provided, which can be used for targeted fluorescence imaging of tumors, surgical navigation, tumor metastasis monitoring, dual-targeted drug conjugate development, etc., which comprises the dual-targeted fluorescent probe described in the fifth aspect of the present invention, and, optionally, one or more pharmaceutically acceptable excipients.

[0260] In the eighth aspect of the present invention, a dual-targeting anti-tumor drug molecule is provided, which includes a recognition group, a drug residue and a linker, wherein the recognition group and the drug residue are covalently linked via the linker, and the recognition group includes hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue) and a glucose residue.

[0261] Specifically, the hyaluronic acid or its oligosaccharide residue (especially hyaluronic acid oligosaccharide residue), glucose residue, and connecting group are as described in the fourth aspect of the present invention.

[0262] Specifically, the dual-targeting anti-tumor drug molecule has the following structure:

[0263]

[0264] Wherein, n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20);

[0265] a, b, c, d, e are independently selected from integers of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0266] M is H or an alkali metal (e.g., Na, K);

[0267] L' is an optional suitable group;

[0268] D is the drug residue.

[0269] Specifically, the drug is an anti-tumor drug, for example, cytotoxic antibiotics, plant alkaloids, antimetabolites, alkylating agents, platinum compounds, protein kinase inhibitors, immune checkpoint regulators, etc.

[0270] Specifically, the cytotoxic antibiotics include but are not limited to: ixabepilone, mitomycin, plicamycin, bleomycin, pyrantelone, amrubicin, valrubicin, pirarubicin, mitoxantrone, idarubicin, daunorubicin, aclarubicin, epirubicin, daunorubicin, doxorubicin and dactinomycin, etc.

[0271] Specifically, the plant alkaloids include but are not limited to: trabectedin, cabazitaxel, paclitaxel polyaniline, docetaxel, paclitaxel, demeclocycline, teniposide, etoposide, vinforin, vinflunine, vinorelbine, vindesine, vincristine and vinblastine.

[0272] Specifically, the antimetabolites include but are not limited to: floxuridine, trifluridine, tegafur, fluorouracil, decitabine, azacitidine, capecitabine, gemcitabine, carmofur, cytarabine, nelarabine, clofarabine, fludarabine, cladribine, thioguanine, mercaptopurine, pralatrexate, pemetrexed, raltitrexed and methotrexate, etc.

[0273] Specifically, the alkylating agent includes but is not limited to: dacarbazine, temozolomide, pipobromide, mitropromol, etoside, uracil mustard, ranimustine, nimustine, flutemustine, streptozotocin, semustine, lomustine, carmustine, carboquinone, triazinequinone, thiotepa, mannosulfuron, trothiosulfuron, busulfan, bendamustine, prednimustine, trofosfamide, ifosfamide, methylchlorambucil, melphalan, chlorambucil and cyclophosphamide, etc.

[0274] Specifically, the platinum compound includes but is not limited to: cisplatin, carboplatin, oxaliplatin, satraplatin and polyplatin.

[0275] Specifically, the protein kinase inhibitors include but are not limited to: BTK inhibitors, PI3K inhibitors, SYK inhibitors, JAK inhibitors and the like.

[0276] Specifically, the immune checkpoint regulators include but are not limited to: PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, OX-40 agonists, CD137 agonists, LAG-3 inhibitors, and IDO inhibitors.

[0277] In some embodiments, the PD-1 inhibitor is selected from nivolumab, pidilizumab, pembrolizumab, AMP-224 (CAS#1422184-00-6), STI-A1110, AMP-514 (MEDI0680, CAS#1642374-69-3), TSR-043 and AUNP-12 (AUR-012, Aurigene-012, Aurigene NP-12).

[0278] In some embodiments, the PD-L1 inhibitor is selected from RG 7446, BMS-936559 (MDX1105, CAS# 1422185-22-5), MSB0010718C, STI-A1010, avelumab, atezolizumab, and durvalumab.

[0279] In some embodiments, the CTLA-4 inhibitor is selected from ipilimumab and tremelimumab.

[0280] In some embodiments, the OX40 agonist is selected from anti-OX40, TIM3 antibodies, and Immutune IMP701.

[0281] In some embodiments, the CD137 agonist is selected from urelumab and utomilumab.

[0282] In some embodiments, the LAG-3 inhibitor is BMS-986016 (CAS #1683572-29-3).

[0283] In some embodiments, the IDO inhibitor is selected from GDC-0919 (CAS#1402836-58-1), indoximod, 1-methyl-D-tryptophan (NSC-721782), NLG919 (CAS#1402836-58-1) epacadostat, and norharman.

[0284] In the ninth aspect of the present invention, a pharmaceutical composition is provided, comprising the dual-targeting anti-tumor drug molecule described in the eighth aspect of the present invention, and, optionally, one or more pharmaceutically acceptable excipients.

[0285] The HA-glucose dual-targeting molecule prepared by the present invention can simultaneously target the GLUT and CD44 receptors of tumors (e.g., lung tumors) by utilizing the interaction between HA-CD44 and Glc-GLUT1. The dual-targeting molecule can deliver fluorescent molecules into (e.g., lung tumor) cells in a targeted manner, reduce the aggregation of fluorescent molecules in normal cells, and at the same time improve the tumor cell endocytosis efficiency of fluorescent labeled molecules.

[0286] The chemical structures of the two targeting molecules HA and glucose in the dual-targeting probe molecules prepared by the present invention are clear, and the covalent bond connection sites and compositions are clear. The fluorescent molecules can simulate the connection between drugs and the dual-targeting molecules, which is beneficial to the subsequent drug development using dual-targeting molecules coupled to drugs. The dual-targeting probe molecules have very good research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0287] Figure 1 The figure shows the synthesis route of the fluorescently labeled HA12-glucose dual-targeting molecule (compound 28), wherein building blocks 12, 18, and 25 are first synthesized by chemical synthesis, and then the dual-targeting molecule is obtained by click reaction.

[0288] Figure 2 Shown is the synthetic route of fluorescently labeled HA12 sugar (compound 29).

[0289] Figure 3 Shown is the synthetic route of fluorescently labeled triethylene glycol-arm glucose (compound 20).

[0290] Figure 4 Shown is a diagram of the enzymatic synthesis of HA12.

[0291] Figure 5The results of the A549 cell uptake test of fluorescent probes are shown, where HGR: HA / Glc dual-targeting fluorescent probe (Compound 28); HR: HA single-targeting fluorescent probe (Compound 29); GR: Glc single-targeting fluorescent probe (Compound 20). Scale bar: 50 μm.

[0292] Figure 6 The results of quantitative analysis of fluorescence intensity uptake by A549 cells are shown, where HGR: HA / Glc dual-targeting fluorescent probe (Compound 28); HR: HA single-targeting fluorescent probe (Compound 29); GR: Glc single-targeting fluorescent probe (Compound 20). (n=4, mean±sd, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0293] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0294] In the present invention, the term "alkyl" refers to a straight or branched hydrocarbon chain radical that does not contain an unsaturated bond, and the hydrocarbon chain radical is connected to the rest of the molecule by a single bond. In this article, C1-6 alkyl refers to a group containing 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc.

[0295] In the present invention, there is a description of "protecting group". When a multifunctional organic compound is reacted, in order to make the reaction occur only at the desired group and avoid other groups from being affected, other groups are protected before the reaction and restored after the reaction is completed. In order to protect the group, the substituent introduced on the group is called "protecting group". For example, "hydroxy protecting group" refers to a substituent connected to a hydroxyl group that prevents or protects the hydroxyl functional group. Exemplary hydroxy protecting groups include but are not limited to alkyl ethers or substituted alkyl ethers such as methyl, allyl, benzyl, substituted benzyl, silyl ethers such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), t-butyldimethylsilyl (TBDMS), etc., acetals or ketals such as tetrahydropyran (THP), esters such as formates, acetates, chloroacetates, dichloroacetates, trichloroacetates, trifluoroacetates, methoxyacetates, etc., carbonates, sulfonates such as methanesulfonates (mesylate), benzylsulfonate. Exemplary amino protecting groups include, but are not limited to, alkyl or substituted alkyl, such as methyl, allyl, benzyl, substituted benzyl, amides, such as acetyl, carbamates, such as Boc, sulfonates such as mesylate (mesylate), benzyl sulfonate and toluene sulfonate (Ts). For a general description of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991. A reagent that can protect a certain group is called a protecting agent for the group. For example, (Boc)2O can be used as a protecting agent for amino groups, and the corresponding amino protecting group is Boc; for example, MsCl can be used as a protecting agent for hydroxy groups, and the corresponding hydroxy protecting group is Ms.

[0296] Immune checkpoint proteins are components of the immune system, which can generally be used to stimulate or inhibit immune signals (e.g., T cell activation signals). Non-limiting examples of stimulatory immune checkpoint proteins include CD27, CD40, OX40, GITR, CD137, CD28, HVEM, and ICOS. Non-limiting examples of inhibitory immune checkpoint proteins include adenosine A2A receptors, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, TIM-3, and VISTA (C10 or f54).

[0297] Immune checkpoint modulatory compounds are any compounds that modulate the activity of immune checkpoint proteins, such as antibodies, small molecules, biologics, or polysaccharides. Regulation of immune checkpoint proteins can include the activity of compounds as agonists, antagonists, allosteric effectors, or any effect from binding to immune checkpoint proteins or protein ligands that changes the conventional biological function of immune checkpoint proteins. Many tumors express high levels of inhibitory immune checkpoint proteins in order to circumvent detection by T cells and other immune system components. Blocking inhibitory immune checkpoint proteins (such as PD-1) using antagonist antibodies enhances the immune response to detect and destroy tumor cells. Conversely, activating stimulatory immune checkpoint proteins (such as OX-40) using agonist antibodies enhances the immune response to recognize and destroy tumor cells.

[0298]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0299] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0300] Example 1: Preparation of Compound 5

[0301] The synthetic route is as follows:

[0302]

[0303] Potassium tert-butoxide (7.47 g, 66.6 mmol) was dissolved in 300 mL of tetrahydrofuran, triethylene glycol (compound 1, 20 g, 133.2 mmol) was added under argon protection in an ice bath, and stirred at room temperature for 30 min, which was component 1; propargyl bromide (7.92 g, 66.6 mmol) was dissolved in 50 mL of tetrahydrofuran, which was component 2. Component 2 was added to component 1, and the reaction was stirred at room temperature for 14 h. After the reaction was completed, the solvent was evaporated and purified by column chromatography (the mobile phase was pure ethyl acetate) to obtain compound 2 (7.2 g, 57%).

[0304] Compound 2 (7 g, 37.19 mmol) was added to a round-bottom flask, 42 ​​mL of dry dichloromethane was added to dissolve, and pyridine (5.99 mL, 74.4 mmol) was added, and p-toluenesulfonyl chloride (10.64 g, 55.8 mmol) was added under ice bath conditions, and the mixture was stirred and reacted for 12 h after warming to room temperature. After the reaction was completed by monitoring by TLC (PE: AcOEt = 1: 1), dichloromethane (100 mL) and hydrochloric acid solution (100 mL, 3%) were added to the reaction solution for extraction, and then the organic phase was extracted by adding saturated sodium chloride solution (100 mL), and the organic phase was dried over anhydrous sodium sulfate, the desiccant was filtered off, the filtrate was evaporated, and purified by column chromatography (mobile phase PE: AcOEt = 2.5: 1) to obtain compound 3 (5.15 g, 40%). 1 H NMR(400MHz, CDCl3)δ:7.71(dd,J=8.2,1.6Hz,2H),7.27(d,J=8.0Hz,2H),4.10(t,J=1.9Hz,2H),4.07(td,J=4.7,1.4Hz,2H) ,3.60(dddd,J=6.6,5.2,3.4,1.4Hz,5H),3.56(dt,J=7.2,2.6Hz,2H),3.50(d,J=1.5Hz,5H),2.40–2.37(m,1H),2.36(s,3H).

[0305] Compound 3 (5.0 g, 14.6 mmol) was added to a round-bottom flask, 49 mL of N,N-dimethylformamide was added to dissolve, and then methyl 3,5-dihydroxybenzoate (0.82 g, 4.9 mmol) and potassium carbonate (6.7 g, 48.7 mmol) were added, and the mixture was stirred at 80°C for 18 h. After the reaction was completed, the mixture was cooled to room temperature by TLC (PE: AcOEt = 1:3), and extracted with ethyl acetate (300 mL) and water (300 mL). The organic phase was washed once with a saturated sodium chloride solution (200 mL), and the organic phase was dried over anhydrous sodium sulfate, the desiccant was filtered out, the filtrate was evaporated, and the mixture was purified by column chromatography (mobile phase PE: AcOEt = 1:2) to obtain compound 4 (2.5 g, 83%). 1 H NMR(600MHz, CDCl3)δ:7.18(d,J=2.3Hz,2H),6.69(t,J=2.3Hz,1H),4.19(d,J=2.4Hz,4H),4.15–4.11 (m,4H),3.88(s,3H),3.87–3.83(m,4H),3.75–3.70(m,5H),3.70–3.66(m,12H),2.42(t,J=2.4Hz,2H); 13C NMR (150MHz, CDCl3) δ: 166.77, 159.77, 131.89, 108.03, 106.94, 79.67, 74.56, 70.85, 70.68, 70.48, 69.61, 69.12, 67.75, 58.41, 52.23.

[0306] Compound 4 (1 g, 1.97 mmol) was added into a round-bottom flask, and 47 mL of anhydrous ethanol was added to dissolve it. Potassium hydroxide (4 mL, 1 M) was then added and stirred at 80°C for 2 h under reflux. After the reaction was completed as monitored by TLC (PE: AcOEt = 1:3), an acidic cationic resin was added to adjust the pH of the reaction solution to neutral, the resin was filtered out, and the reaction solution was evaporated to obtain compound 5 (0.97 g, 100%). 1 HNMR (400MHz, CDCl3) δ: 7.10 (d, J = 2.3Hz, 2H), 6.48 (t, J = 2.3Hz, 1H), 4.15 (d, J = 2.4Hz, 4H), 4.0 2(dd,J=5.9,3.5Hz,4H),3.72(dd,J=5.7,3.5Hz,5H),3.69–3.55(m,18H),2.45(t,J=2.4Hz,2H). 13 C NMR (100MHz, CDCl3) δ: 171.48, 159.02, 138.56, 107.92, 104.47, 79.62, 74.87, 70.41, 70.33, 70.17, 69.65, 68.83, 67.24, 58.27.

[0307] Example 2: Preparation of Compound 18

[0308] The synthetic route is as follows:

[0309]

[0310] Note: Indicates that the configuration of the related compound may be α or β configuration.

[0311] Weigh glucose (50g, 277.5mmol) in a 500mL round-bottom flask, add acetic anhydride (200mL) to dissolve, then add anhydrous sodium acetate (40g, 487.6mmol), stir evenly and slowly heat to 120℃, react under reflux for about 2h. After the reaction is completed by monitoring by TLC (PE: AcOEt = 1:2), ice water is added to the reaction solution to quench the reaction until a white solid is precipitated, and the solid is obtained by suction filtration in a Buchner funnel and dried in an oven. The filtrate is extracted with ethyl acetate, and the organic phase is added with sodium bicarbonate solution to remove acid, and then anhydrous sodium sulfate is added to dry, and the desiccant is filtered off and evaporated to dryness to obtain compound 14 (110g, 100%).

[0312] Compound 14 (40 g, 103 mmol) and ammonium acetate (31.6 g, 410 mmol) were added to a round-bottom flask, and 75 mL of methanol and 75 mL of tetrahydrofuran were added to dissolve the mixture. The mixture was reacted overnight at room temperature. After the reaction was completed, the reaction solution was evaporated to dryness using a rotary evaporator and purified using column chromatography (mobile phase PE: AcOEt = 1:1) to obtain a / b mixture 15 (22 g, 60%).

[0313] Compound 15 (16.5 g, 47.2 mmol) was added to a round-bottom flask, 50 mL of dry dichloromethane was added to dissolve, and trichloroacetonitrile (14.2 mL, 141.6 mmol) and 1,8-diazabicycloundec-7-ene (700 μL, 4.72 mmol) were added, and the mixture was stirred at room temperature for about 4 h. After the reaction was completed, the reaction solution was evaporated and purified by column chromatography (mobile phase PE: AcOEt = 4: 1) to obtain compound 16 (16.8 g, 72%). 1 H NMR (600MHz, CDCl3) δ: 8.69 (s, 1H), 6.55 (d, J = 3.7Hz, 1H), 5.56 (t, J = 9.8Hz, 1H), 5.17 (dd, J = 10.3, 9.5Hz, 1H), 5.13 (dd, J = 10.2, 3 .7Hz,1H),4.27(dd,J=12.4,4.2Hz,1H),4.23–4.18(m,1H),4.16–4.08(m,1H),2.07(s,3H),2.04(s,3H),2.02(s,3H),2.01(s,3H). 13 C NMR (150MHz, CDCl3) δ: 170.56, 170.00, 169.85, 169.50, 160.80, 92.91, 90.69, 70.02, 69.88, 69.73, 67.79, 61.38, 60.39, 20.67, 20.59, 20.44.

[0314] Compound 16 (2.33 g, 4.74 mmol) was added to a round-bottom flask, 20 mL of dried dichloromethane was added to dissolve, and then compound 10 (1.2 g, 7.11 mmol) and trifluoromethanesulfonic acid (70 mg, 0.5 mmol) were added, and the mixture was stirred and reacted overnight under argon protection in an ice bath. After the reaction was completed, triethylamine was added to quench the reaction, and the reaction solution was evaporated to dryness and purified by column chromatography (PE: AcOEt = 3.5: 1) to obtain compound 17 (1 g, 42%). 1 H NMR (400MHz, CDCl3) δ: 5.18 (t, J=9.5Hz, 1H), 5.06 (dd, J=10.0, 9.4Hz, 1H), 4.97 (dd,J=9.7,8.0Hz,1H),4.59(d,J=8.0Hz,1H),4.23(dd,J=12.3,4.8Hz,1H),4.11 (dd,J=12.3,2.4Hz,1H),3.92(ddd,J=11.1,4.9,3.8Hz,1H),3.76–3.58(m,10H), 3.38(dd,J=5.6,4.5Hz,2H),2.06(s,3H),2.02(s,3H),2.00(s,3H),1.98(s,3H). 13 C NMR (100MHz, CDCl3) δ: 170.67, 170.28, 169.43, 169.36, 100.85, 72.84, 71.79, 71.30, 70.73, 70.71, 70.43, 70.05, 69.07, 68.44, 61.98, 50.69.

[0315] Compound 17 (0.4 g, 0.72 mmol) was added to a round-bottom flask, 2 mL of methanol was added to dissolve, and then an appropriate amount of sodium methoxide was added to adjust the pH of the reaction solution to 9, and the reaction was carried out at room temperature for 4 h. After the reaction was completed by monitoring by TLC (PE: AcOEt = 1:3), an acidic cationic resin was added to adjust the pH of the reaction solution to neutral, and the filtrate was filtered with diatomaceous earth and evaporated to dryness to obtain compound 18 (0.27 g, 97.2%).

[0316] Example 3: Preparation of Compound 12

[0317]

[0318]

[0319] Add 1.4L of anhydrous methanol to a round-bottom flask, put it in a -20℃ refrigerator and completely cool it. Then take it out and add 12mL of acetyl chloride. Stir slowly at 4℃. Add 10g of oligosaccharide mixture 6 to this solution and continue stirring at 4℃ for 3 days. After the solution changes from light yellow to dark red, add Na2CO3 to adjust the pH to neutral, filter out the precipitate, and evaporate the solvent to obtain a dark red solid.

[0320] The red solid obtained in the previous step was added to a round-bottom flask, and 83.2 mL of pyridine was added. Under argon protection at 0°C, 41.6 mL of acetic anhydride was added dropwise to the above mixture within 30 min, and the mixture was slowly heated to room temperature and stirred for 20 h. TLC monitoring (CH2Cl2 / EtOAc / MeOH, 1:1:0.2) was performed after the reaction was completed. The reaction system was cooled to 0°C, and 16.8 mL of methanol was added and stirred for 20 min to quench the reaction. The solvent was evaporated, and 320 mL of EtOAc was added to dilute and continued to stir for 20 min. The precipitate was filtered off, the filtrate was evaporated, and the mixture was purified by column chromatography (CH2Cl2 / EtOAc / MeOH, 1:1:0.08-1:1:0.12, containing 0.1% Et3N) to obtain compound 7, which was a mixture of α / β configurations (1.2 g). 1 H NMR(400MHz, DMSO-d6)δ:7.84(d,J=9.2Hz,2H),7.75(d,J=9.0Hz,1H),7.43(d,J=9.3Hz,3H),5.77(d,J=3.7Hz,2H),5.54(d,J =8.3Hz,1H),5.27(t,J=9.5Hz,3H),5.06(t,J=9.3Hz,1H),4.97(dt,J=9.3,5.1Hz,4H),4.91–4.78(m,9H),4.70(t,J=9.3Hz,1H ),4.66–4.47(m,8H),4.30(d,J=10.0Hz,6H),4.22(dd,J=12.6,4.0Hz,3H),4.14–3.99(m,9H),3.98–3.89(m,6H),3.89–3.78(m ,19H),3.76–3.68(m,4H),3.64(s,9H),3.51(s,3H),2.18(s,6H),2.05–1.95(m,50H),1.95–1.89(m,33H),1.89–1.84(m,19H).

[0321] Compound 7 (3 g, 2.4 mmol) was dissolved in 30 mL THF under argon protection, and DMAPA (615 μL, 4.9 mmol) was added in 5 times, each time with an interval of 30 min. The reaction was carried out at room temperature for 1 h. After the reaction was completed by monitoring by TLC (CH2Cl2 / EtOAc / MeOH, 1:1:0.2), 1 M hydrochloric acid solution was added to quench the reaction, 300 mL CH2Cl2 was added to dilute the reaction solution, and the organic phase was extracted with saturated NaHCO3 solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, the desiccant was filtered out, and the organic phase was evaporated to dryness, and purified by column chromatography (CH2Cl2:EA:MeOH=1:1:0.08-1:1:0.2) to obtain compound 8.

[0322] Compound 8 was dissolved in 30 mL of dry CH2Cl2 solution under ice bath condition, and trichloroacetonitrile (1.9 mL, 19.2 mmol) and DBU (72 μL, 0.48 mmol) were added. The reaction was carried out at room temperature under argon protection for 2 h. After the reaction was completed, the solvent was evaporated and purified by column chromatography (CH2Cl2:EA:MeOH=1:1:0.07-1:1:0.1) to obtain compound 9 (1.25 g, 39%, from compound 7 to 9). 1 H NMR (400MHz, CDCl3) δ: 8.80 (s, 1H), 6.21 (d, J = 3.8Hz, 1H), 5.90 (d, J = 7.7Hz, 1H), 5.81 (d, J = 9.6Hz, 1H), 5.19 (t, J=9.3Hz,1H),5.14–5.04(m,3H),4.90–4.86(m,1H),4.81–4.72(m,4H),4.64–4.55(m,2H),4.39(t,J=9.6Hz,1H), 4.27(dd,J=12.4,4.4Hz,1H),4.16(dd,J=12.4,4.3Hz,1H),4.13–3.99(m,6H),3.96(d,J=10.0Hz,1H),3.91–3.8 3(m,1H),3.82(s,3H),3.70(s,3H),3.63(ddd,J=9.9,4.4,2.4Hz,1H),3.16(d,J=9.0Hz,1H),2.08–1.96(m,40H). 13C NMR(100MHz, CDCl3)δ:171.20,171.01,170.75,170.16,169.86,169.78,169.70,169.33,169.1 6,169.09,167.95,166.97,160.17,100.85,99.97,98.78,95.28,90.83,75.77,74.80,72.26,7 2.10,71.81,71.76,71.59,70.13,69.37,68.00,67.72,61.76,61.65,60.40,57.55,53.43,53.04,52.73,51.81,23.56,23.41,23.19,21.05,20.77,20.71,20.69,20.57,20.54,20.49,20.43.

[0323] Compounds 9 (1.22 g, 0.92 mmol) and 10 (1.45 g, 8.27 mmol) were dissolved in 15 mL of dry CH2Cl2 solution, trifluoromethanesulfonic acid (8.8 μL, 0.1 mmol) was added, and the mixture was reacted at room temperature overnight under argon protection. After the reaction was completed, the solvent was evaporated and purified by column chromatography (CH2Cl2:EA:MeOH=1:1:0.07-1:1:0.1) to obtain compound 11 (945 mg, 77%). 1H NMR(400MHz, CDCl3)δ:6.28–6.22(m,1H),6.03(d,J=7.3Hz,1H),5.17(t,J=9.3Hz,1H),5.09(t,J=9.6Hz,1H),5.04(t,J=8.7Hz,1H),4 .85(q,J=8.8Hz,3H),4.79(t,J=10.0Hz,4H),4.66(d,J=7.9Hz,1H),4.58(d,J=8.0Hz,1H),4.50(t,J=9.9Hz,1H),4.30(dd,J=12.6,4. 5Hz,1H),4.25(t,J=9.5Hz,1H),4.17(dd,J=12.3,5.0Hz,1H),4.06(dt,J=24.3,9.8Hz,2H),3.96(t,J=11.1Hz,3H),3.89(d,J=11.7Hz ,1H),3.79(s,4H),3.75–3.65(m,17H),3.61(d,J=8.7Hz,9H),3.43(qt,J=13.0,5.7Hz,3H),2.99(q,J=8.7Hz,1H),2.17–1.91(m,50H). 13 C NMR(100MHz, CDCl3)δ:170.74,170.18,169.84,169.52,169.44,167.94,167.05, 100.81,100.29,100.17,98.48,78.26,76.73,75.87,74.60,72.36,72.32,71.83, 71.79,71.63,70.92,70.60,70.42,69.93,69.51,68.87,68.73,68.19,62.53,62.09,58.18,56.96,53.05,52.80,50.57,23.62,20.90,20.83,20.67,20.61,20.54.

[0324] Compound 11 (400 mg, 0.3 mmol) was dissolved in 4.8 mL THF / H2O (3:1), and freshly prepared 0.75 mL hydrogen peroxide solution (30 wt% aqueous solution) and 1.5 mL LiOH (1 M) were added under ice bath conditions. The reaction was continued at 0°C for 1 hour and then at room temperature for 15 hours. The reaction system was cooled to 0°C, 1.5 mL methanol and 3.75 mL NaOH solution (4 M) were added, and the reaction was continued at room temperature for 10 hours. After the reaction was completed, acidic cationic resin was added to adjust the pH to 3, the resin was filtered off, the solvent was evaporated, and compound 12 (235 mg, 83%) was purified by P2 gel exclusion chromatography.1 H NMR (600MHz, CDCl3) δ: 4.50 (dd, J=8.5, 6.2Hz, 2H), 3.93 (ddd, J=11.8, 5.7, 3.0Hz, 1H), 3.86 (dd, J=5.1, 2.2Hz, 1H), 3.84 (dd, J=5.1, 2.1Hz,1H),3.78(ddd,J=10.3,8.5,2.9Hz,2H),3.73–3.59(m,17H),3.54–3.37(m,9H),3.32–3.23(m,2H),1.96(s,3H),1.95(s,3H). 13 C NMR(150MHz, CDCl3)δ:175.47,174.92,174.62,174.15,103.09,102.96,100.82,100.56,82.99,82.32,79.97,76.37,75.71,75.39,7 5.30,73.59,72.70,72.43,71.69,69.69,69.64,69.57,69.21,69.03,68.58,68.52,60.69,60.52,54.46,54.23,50.12,22.49,22.23.

[0325] Example 4: Preparation of Compound 27

[0326]

[0327] The modular assembly strategy of HA oligosaccharides uses compound 12 as the starting substrate and gradually adds sugar nucleotides and GA-T and GN-T (e.g. Figure 4 As shown), HA12 oligosaccharide is prepared (reference, for example, Li S, Wang S, Qu J, Han J, Yang L, LiY, Li L, Jia Q, Chen C, Ling P, Fang J. 2023. Programmable one cycle–onedisaccharide unit modular synthesis of hyaluronan and chondroitin hybrid glycans[J]. Green Chemistry, 25(5): 2021-2027.) (HA12 oligosaccharide can better enter tumor cells and has better targeting effect. It is used as an example of HA oligosaccharide in the embodiment to verify the effect of the dual-targeting molecule of the present invention).

[0328] The reaction system is shown in Table 1:

[0329] Table 1 HA oligosaccharide synthesis system

[0330]

[0331]

[0332] Table 2 Biological enzymes used

[0333]

[0334] The reaction was carried out at 30°C for 12 hours, and the reaction was monitored by TLC (EA:MeOH:H2O:HAC=5:2:3:1) and ESI-MS. After the reaction, the mixture was boiled at 100°C for 10 minutes. The protein was removed by centrifugation at 12,000 rpm for 10 minutes. The target product was separated and purified by Bio-rad P6 gel separation.

[0335] Four GlcNAc-GlcA disaccharide repeating units were extended at the non-reducing end of compound 12 by a four-step enzymatic cycle reaction to obtain compound 27. LCMS (ESI + )Calcd for C 90 H 133 N9O 69 [M-5H] 5- :488.95,Found:488.94.

[0336] Example 5: Preparation of Compound 25

[0337]

[0338] Rhodamine B (21, 2 g, 4.18 mmol) and compound 22 (693 mg, 2.79 mmol) were dissolved in 50 mL CH2Cl2, and DIPEA (1.47 mL), EDCI (803 mg, 4.19 mmol) and catalytic amount of DMAP were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the solvent was evaporated and purified by column chromatography (EA:PE = 1:1-3:1) to obtain compound 23 (609 mg, 32%). 1H NMR(600MHz, CDCl3)δ:7.91–7.86(m,1H),7.44–7.36(m,2H),7.08–7.01(m, 1H),6.41(d,J=8.9Hz,2H),6.36(d,J=2.6Hz,2H),6.25(dd,J=8.9,2.6Hz,2H ),5.08(t,J=5.8Hz,1H),3.49–3.41(m,4H),3.39–3.28(m,12H),3.24(m,2H) ,3.17(t,J=7.3Hz,2H),1.40(s,9H),1.16(s,3H),1.15(s,6H),1.13(s,3H). 13 C NMR(150MHz, CDCl3)δ:168.25,156.03,153.73,153.26,148.75,132.38,130.96,128.82,127.95,123.76,12 2.74,108.05,105.51,97.78,79.01,70.23,70.17,69.84,67.87,64.84,44.34,40.33,39.24,28.40,12.59.

[0339] Compound 23 (300 mg, 0.44 mmol) was dissolved in 3 mL CH2Cl2, and 1.5 mL trifluoroacetic acid was added to react at room temperature for 4 h. After the reaction was completed by TLC monitoring, CH2Cl2 was added to dilute the reaction solution, and the mixture was extracted with saturated Na2CO3 solution. The organic phase was dried over anhydrous sodium sulfate, the desiccant was filtered out, and the filtrate was evaporated to dryness to obtain compound 24 (230 mg, 89%).

[0340] Compound 24 (260 mg, 0.5 mmol) and 5 (149 mg, 0.3 mmol) were dissolved in 4 mL CH2Cl2, and DIPEA (1.47 mL), EDCI (803 mg, 4.19 mmol) and catalytic amount of DMAP were added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the solvent was evaporated and purified by column chromatography (EA:PE=1:1-3:1) to obtain compound 25 (210 mg, 66%).

[0341] Example 6: Preparation of Compound 28

[0342]

[0343] Compound 18 (44 mg, 0.13 mmol) and 25 (550 mg, 0.52 mmol) were dissolved in a mixture of 1.5 mL CH2Cl2, 0.5 mL water and 1.5 mL MeOH, and freshly prepared CuSO4 solution (135 μL, 0.2 M) and ascorbic acid solution (675 μL, 0.2 M) were added. The mixture was reacted at room temperature for 24 h. After the reaction was completed, the solvent was evaporated and purified by column chromatography (EA: MeOH = 20: 1-4: 1) to obtain compound 26 (48 mg, 27%). LCMS (ESI + )Calcd for C 10 H 19 N3NaO7[M+Na] + :316.11,Found:316.1114.

[0344] Compound 26 (48 mg, 0.035 mmol) and 27 (14 mg, 0.006 mmol) were dissolved in a mixture of 1.5 mL of water and 1.5 mL of MeOH, and freshly prepared CuSO4 solution (30 μL, 0.2 M) and ascorbic acid solution (150 μL, 0.2 M) were added, and the mixture was reacted at room temperature for 48 h. After the reaction was completed, the solvent was evaporated and the product was purified by alternating Bio-rad P2 gel and DEAE ion exchange resin to obtain compound 28 (10 mg, 43%). LCMS (ESI + )Calcd for C 161 H 235 N 16 O 90 [M-4H] 3- :1277.81,Found:1277.78.

[0345] Example 7: Preparation of Compound 29

[0346]

[0347] Compound 27 (10 mg, 0.004 mmol) and 19 (6 mg, 0.012 mmol) were dissolved in a mixture of 1.5 mL of water and 1.5 mL of MeOH, and freshly prepared CuSO4 solution (20 μL, 0.2 M) and ascorbic acid solution (100 μL, 0.2 M) were added. The mixture was reacted at room temperature for 48 h. After the reaction was completed, the solvent was evaporated and the product was purified by alternating Bio-rad P2 gel and DEAE ion exchange resin to obtain compound 29 (10 mg, 43%). LCMS (ESI+ )Calcd for C 121 H 167 N 12 O 71 [M-4H] 5- :585.00,Found:584.99.

[0348] Example 8: Preparation of Compound 20

[0349]

[0350] Compounds 18 (20 mg, 0.06 mmol) and 19 (29 mg, 0.06 mmol) were dissolved in a mixture of 255 μL methanol, 85 μL water and 255 μL CH2Cl2, and freshly prepared CuSO4 solution (15 μL, 0.2 M) and ascorbic acid solution (75 μL, 0.2 M) were added. The mixture was reacted at room temperature for 24 h. After the reaction was completed, the solvent was evaporated and purified by column chromatography (EA: MeOH = 20: 1-4: 1) to obtain compound 20 (20 mg, 41%). LCMS (ESI + )Calcd for C 43 H 57 N6O 10 [M] + :817.41,Found:817.4166.

[0351] Example 9: Cellular uptake detection of dual-targeting and single-targeting fluorescent probes

[0352] 1x10 cells were seeded in a confocal laser culture dish. 5 A549 cells (human non-small cell lung tumor cells) were cultured overnight at 37°C and 5% CO2, and then the culture medium was discarded. Fresh culture medium and a final concentration of 25 μM HA / Glc dual-targeting fluorescent probe compound 28, HA single-targeting fluorescent probe compound 29 or Glc single-targeting fluorescent probe compound 20 were added and incubated in the dark for 4 h. The culture medium was discarded, and the cells were gently washed three times with PBS. 4% paraformaldehyde was added to fix the cells at 4°C for 15 min, the fixative was discarded, and the cells were gently washed three times with PBS. 10 μg / mL DAPI solution was added and incubated at 37°C for 15 min, the DAPI solution was discarded, and the cells were gently washed three times with PBS. The cells were observed and photographed under a laser confocal microscope (LSM 900, Zeiss, Germany). Figure 5 and 6As shown in the figure, after 4 hours of incubation, in A549 cells, there was a significant difference between the dual-targeted fluorescent probe and the two single-targeted fluorescent probes in entering the tumor cells. The HA / Glc dual-targeted fluorescent probe entered the tumor cells more, 4 times and 32 times that of the HA single-targeted fluorescent probe and the Glc single-targeted fluorescent probe, respectively. It is speculated that this is probably because the HA / Glc dual-targeted fluorescent probe can enter cells through the interaction of HA-CD44 and enter tumor cells through the interaction of Glc-GLUT1, while the HA single-targeted fluorescent probe and the Glc single-targeted fluorescent probe can only enter tumor cells through the interaction of HA-CD44 and Glc-GLUT1, respectively. This experimental result shows that the HA / Glc dual-targeted fluorescent probe has a significant advantage in tumor cell uptake efficiency compared with the single-targeted fluorescent probe, and it is expected to be efficiently used in targeted fluorescence imaging of tumor cells and tissues, surgical navigation, tumor metastasis monitoring, and efficacy evaluation.

[0353] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0354] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0355] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A preparation method based on hyaluronic acid and glucose dual-targeting molecules, comprising the following steps: preparing intermediate M1 from hyaluronic acid oligosaccharide, preparing intermediate M2 from glucose, preparing intermediate M3 from the target compound, and then performing click reaction between M1, M2 and M3 to obtain the target product; in, n is an integer from 1 to 10; a, b, c, d, e are independently selected from integers of 1-10; M is H or an alkali metal; R1 to R6 are independently selected from: H, C1-6 alkyl; The preparation method comprises the following reaction route: （Ⅴ）。 2. The method according to claim 1, characterized in that The click reaction is carried out in the presence of a copper catalyst.

3. The method according to claim 1, characterized in that The click reaction system also includes a reducing agent.

4. The method according to claim 3, characterized in that The reducing agent is ascorbic acid.

5. The method according to claim 1, characterized in that The click reaction is carried out in the presence of copper sulfate and ascorbic acid.

6. The method according to claim 1, characterized in that The click reaction temperature is 20-30°C.

7. The method according to any one of claims 1 to 6, characterized in that: The preparation of M1 includes the following reaction route: Wherein, R7 is C1-6 alkyl; R8 is a hydroxyl protecting group; Indicates that step (B6) is optional.

8. The method according to claim 7, characterized in that The step (B1) comprises: reacting compound 6 with alcohol and hydroxyl protecting agent respectively to obtain compound 7; The step (B2) comprises: mixing compound 7 with DMAPA, and reacting under the protection of an inert gas to obtain compound 8; The step (B3) comprises: mixing compound 8 with trichloroacetonitrile and a base, and reacting under the protection of an inert gas to obtain compound 9; The step (B4) comprises: mixing compound 9 and compound The mixture is mixed and reacted in the presence of an acid under the protection of an inert gas to obtain compound 11.

9. The method according to any one of claims 1 to 6, characterized in that: The preparation of M2 includes the following reaction route: Wherein, R9 is a hydroxyl protecting group.

10. The method according to claim 9, characterized in that The step (C1) comprises: reacting compound 13 with a hydroxyl protecting agent to obtain compound 14; The step (C2) comprises: mixing compound 14 with ammonium acetate, reacting to obtain compound 15; The step (C3) comprises: mixing compound 15 with trichloroacetonitrile and a base, and reacting to obtain compound 16; The step (C4) comprises: compound 16 and compound The mixture is mixed and reacted in the presence of an acid under the protection of an inert gas to obtain compound 17.

11. The method according to any one of claims 1 to 6, characterized in that: The preparation of M3 includes the following reaction route: Among them, R 10 It is an amino protecting group.

12. The method according to claim 11, characterized in that The step (D1) comprises: mixing compounds 21 and 22, adding DIPEA, EDCI and DMAP, and reacting to obtain compound 23; The step (D3) comprises: mixing compound 24 and compound 5, adding DIPEA, EDCI and DMAP, and reacting to obtain compound M3.

13. The method according to claim 12, characterized in that The preparation of compound 5 includes the following reaction route: Among them, R 11 is a hydroxyl protecting group; R 12 It is a C1-6 alkyl group.

14. The method according to claim 13, characterized in that The step (E1) comprises: in a solvent E1, mixing compound 1 with propargyl bromide and a base, and reacting to obtain compound 2; The step (E2) comprises: reacting compound 2 with a hydroxyl protecting agent to obtain compound 3; The step (E3) comprises: compound 3 and compound , and a base, and react to obtain compound 4; The step (E4) comprises: mixing compound 4 with alcohol, adding a base, and reacting to obtain compound 5.

15. A dual targeting molecule having the following structure: （Ⅴ）； in, n is an integer from 1 to 10; a, b, c, d, e are independently selected from integers of 1-10; M is H or an alkali metal; R1 to R6 are independently selected from: H, C1-6 alkyl.

16. The dual targeting molecule according to claim 15, characterized in that: The dual-targeting molecule is a dual-targeting fluorescent probe having the following structure: 。 17. A detection reagent or pharmaceutical composition, comprising the dual-targeting molecule according to any one of claims 15-16.

18. The detection reagent or pharmaceutical composition according to claim 17, characterized in that: The detection reagent or pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

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