Tridentate small molecule drug conjugate and preparation method thereof

Abstract

The invention belongs to the technical field of small molecule drug conjugates, and particularly relates to a trident small molecule drug conjugate and a preparation method thereof. The invention aims to provide a novel high-targeting micromolecule drug-coupled drug construction strategy, designs a trident-shaped structure, and introduces three specific targeting ligands to improve the tumor targeting, increase the enrichment of the drug at the tumor site, and reduce the distribution in normal tissues and organs so as to reduce the possibility of causing toxic and side effects of the normal tissues and organs; meanwhile, polyethylene glycol chains with different lengths are introduced, so that the water solubility is improved, and the pharmacokinetic characteristics of pharmacokinetics are improved. The trident-shaped small-molecule coupled drug constructed by the invention has excellent tumor targeting property, can be kept stable in circulation, and effectively releases toxic drug molecules in a special tumor microenvironment to achieve a killing effect. The low-dose medicine has an obvious inhibiting effect on tumor growth and has no obvious side effect on normal organs.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule drug conjugates, and particularly relates to a trident-shaped small molecule drug conjugate and a preparation method thereof. Background Art

[0002] In the field of tumor treatment, although traditional methods such as chemotherapy, targeted therapy and immunotherapy have made significant progress, they still have problems such as limited efficacy and large toxic side effects. Generally, the tumor microenvironment exhibits physiological characteristics that are significantly different from normal tissues, such as hypoxia and high glutathione concentrations. On the other hand, tumor cells have specific or over-expressed substances (for example, antigens, chemical signals, receptors, etc.) on their surfaces, which are not expressed or expressed at low levels in normal cells. Antibody-drug conjugates (ADCs) developed based on these characteristics have brought new breakthroughs in cancer treatment with their unique advantages of being able to specifically recognize tumor cells and accurately deliver cytotoxic drugs. However, the development of ADC drugs also faces many challenges, such as the complexity and high cost of antibody production, immunogenicity, and limited permeability in solid tumors.

[0003] In order to overcome the limitations of ADC drugs, a variety of new drug conjugate forms have emerged in recent years, such as peptide drug conjugates (PDC), small molecule drug conjugates (SMDC), antibody fragment drug conjugates (FDC), etc. Among them, small molecule drug conjugates have received widespread attention due to their unique advantages. Small molecule drug conjugates are usually composed of three parts: small molecule targeting ligands, linkers, and cytotoxic molecules. Compared with antibodies, small molecule targeting ligands have better tissue penetration and diffusion capabilities, can be more evenly distributed inside tumor tissues, and will not aggregate in tumors and other normal cells, thereby reducing toxicity to normal cells. At the same time, a small number of off-target drugs can also be quickly excreted from the body. In addition, the synthesis process of small molecule drug conjugates is relatively simple, low cost, easy to industrialize, and theoretically will not be immunogenic, and has higher safety.

[0004] Despite the numerous potential advantages of small-molecule drug conjugates, their development currently faces several technical challenges. For example, the selection of small-molecule targeting ligands is relatively narrow, making it difficult to obtain targeting ligands with high specificity and affinity. Furthermore, the design of linkers cannot balance stability and cleavability, ensuring drug stability in the bloodstream and effective release of active drugs upon reaching tumor cells. Summary of the Invention

[0005] In response to the deficiencies in the above-mentioned prior art, the present invention provides a trident-shaped small molecule drug conjugate and a method for preparing the same. The trident-shaped small molecule drug conjugate of the present invention is a highly targeted small molecule drug conjugate or a pharmaceutically acceptable salt or a deuterated derivative thereof. The trident-shaped small molecule drug conjugate of the present invention is designed as a trident-shaped structure, and three specific targeting ligands are introduced to improve tumor targeting and increase the enrichment of the trident-shaped small molecule drug conjugate at the tumor site. At the same time, polyethylene glycol chains of different lengths are introduced to balance its water solubility and improve its pharmacokinetic and pharmacokinetic properties. It can be applied to the precision treatment of cancer, and the introduced linker can be cleaved under the reducing environment in the target cell and remain stable in the circulation.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention protects a trident-shaped small molecule drug conjugate, which is composed of a cytotoxic molecule, a linker, a spacer, and three targeting ligands. The cytotoxic molecule is coupled to the linker, and the targeting ligand is coupled to a polyethylene glycol chain. The polyethylene glycol chain and the linker are coupled together through the spacer. The structural formula of the trident-shaped small molecule drug conjugate is as follows:

[0008]

[0009] Among them, n is 6-13640, Represents polyethylene glycol chains of varying lengths.

[0010] D is a cytotoxic molecule, L is a linker, T is a targeting ligand, and the spacer is

[0011] Furthermore, the cytotoxic molecule is selected from one of doxorubicin hydrochloride, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, auristatin, auristatin derivatives, porphyrin, and porphyrin derivatives. The linker is selected from a peptide linker or a disulfide linker, and the peptide linker is selected from any one of valine-citrulline, phenylalanine-lysine, or valine-alanine. The linker can be cleaved by a protease highly or specifically expressed in or around the target cell; a disulfide linker containing a disulfide bond can be cleaved under a reducing environment in the target cell while remaining stable in the circulation. The polyethylene glycol chain is selected from one or more monodisperse polyethylene glycols with 6-48 segments, or one or more polydisperse polyethylene glycols with a molecular weight of 500-600,000. The targeting ligand can specifically bind to cell surface receptors and is a prostate cancer-specific membrane surface antigen targeting molecule and a fibroblast activation protein inhibitor. The targeting ligand is selected from one or more of a prostate cancer-specific membrane surface antigen targeting molecule, folic acid, RGD polypeptide, bombesin polypeptide, octreotide, melanocortin-1 receptor targeting polypeptide, and galactose targeting molecule.

[0012] Furthermore, the present invention prepares a class of highly targeted, fully water-soluble small molecule drug conjugates or pharmaceutically acceptable salts thereof through molecular design, achieving specific delivery of hydrophobic drugs and exhibiting excellent anti-tumor effects in in vitro and in vivo experiments. The structural formula of the trident-shaped small molecule drug conjugate is as follows:

[0013]

[0014]

[0015]

[0016]

[0017] The present invention also protects a method for preparing the above-mentioned trident-shaped small molecule drug conjugate, which comprises the following steps:

[0018] S1. Connecting the cytotoxic molecule and the linker through an amidation reaction to generate a cytotoxic molecule-linker conjugate.

[0019] S2. Connecting the cytotoxic molecule-linker conjugate to the spacer through an amidation reaction to form a cytotoxic molecule-linker-spacer conjugate.

[0020] S3. Connect the cytotoxic molecule-linker-spacer conjugate to three polyethylene glycols through an amidation reaction to form a trident structure.

[0021] S4. Connect the trident-shaped structure to three targeting ligands through an amidation reaction to generate a trident-shaped small molecule drug conjugate.

[0022] The present invention also protects the aforementioned trident-shaped small molecule drug conjugates or their pharmaceutically acceptable salts or deuterated derivatives. Pharmaceutically acceptable salts refer to salt forms formed between the active ingredient of the trident-shaped small molecule drug conjugate and an acid or base, which possess physicochemical properties suitable for pharmaceutical use and have no significant toxic side effects on the human body. Representative salts obtained by acid addition include hydrochloride, phosphate, oxalate, palmitate, lactate, and p-toluenesulfonate. Representative salts obtained by base addition include metal salts and amine salts, such as potassium salt, sodium salt, and calcium salt. Taking the sodium salt as an example, the preparation method is as follows:

[0023] The trident-shaped small molecule drug conjugate (1 equiv) was dissolved in acetone / water (4:1, v / v), and then a 1 mol / L aqueous NaOH solution (1 equiv) was added dropwise. The mixture was stirred at room temperature until completely dissolved (pH 7-8). The acetone was removed by concentration under reduced pressure, and the mixture was freeze-dried to obtain a white crystalline powder, which was a pharmaceutically acceptable salt.

[0024] Deuterated derivatives are modified compounds obtained by replacing the hydrogen atom (H) in a trident-shaped small molecule drug conjugate with its stable isotope deuterium (D). The preparation method is as follows:

[0025] The trident-shaped small molecule drug conjugate is dissolved in deuterated methanol (CD3OD) or D2O, and then a deuterated acid (such as DCl) or a base (such as NaOD) is added. The mixture is heated to reflux (50°C to 80°C) for 24 to 48 hours, and the H / D exchange rate is monitored by NMR. The solvent is concentrated and the mixture is purified by column chromatography to obtain a deuterated derivative.

[0026] The present invention also protects the use of the above-mentioned trident-shaped small molecule drug conjugate or its pharmaceutically acceptable salt or deuterated derivative in cancer treatment of in vivo and in vitro cells and animals, drug delivery, and in the preparation of small molecule drug conjugates.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The purpose of the present invention is to provide a novel strategy for constructing highly targeted small molecule drug conjugates. The structure is designed as a trident, and three specific targeting ligands are introduced to improve tumor targeting, increase the enrichment of the trident small molecule drug conjugate at the tumor site, and reduce its distribution in normal tissues and organs to reduce the possibility of causing toxic side effects in normal organs and tissues. At the same time, polyethylene glycol chains of different lengths are introduced to balance its water solubility and improve its pharmacokinetic properties. The purpose is to solve the problems existing in the ligand selection and linker design of existing small molecule drug conjugates, improve targeting and efficacy, and through the action of targeting ligands, accurately deliver the trident small molecule drug conjugate to the tumor site and specifically release the toxic payload in the special tumor microenvironment to reduce toxic side effects for precise cancer treatment.

[0029] On the one hand, the introduction of three targeting ligands improves the targeting of the trident-shaped small molecule drug conjugate, accurately targets tumor cells, reduces the distribution of the trident-shaped small molecule drug conjugate in non-target tissues, and reduces potential damage to these tissues; on the other hand, when three targeting ligands are introduced, they can simultaneously specifically bind to multiple targets on the surface of tumor cells, producing a multivalent effect. This multivalent binding mode significantly enhances the affinity and binding stability between the trident-shaped small molecule drug conjugate and the target cells.

[0030] The introduction of polyethylene glycol chains significantly improves the water solubility of trifurcated small molecule drug conjugates. The prepared small molecule drug conjugates are completely water-soluble, solving the problem of poor water solubility of most cytotoxic molecules.

[0031] 2. Adoption Figure 3 and Figure 4 Verification has shown that the trident-shaped small molecule drug conjugate constructed in this invention has excellent tumor targeting properties, remains stable in circulation, and effectively releases cytotoxic molecules in the specific tumor microenvironment to achieve a killing effect. Low doses of the trident-shaped small molecule drug conjugate achieve significant inhibition of tumor growth while having no significant side effects on normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the high-resolution mass spectrum of compound A in Example 1.

[0033] Figure 2 The absorption spectrum and fluorescence emission spectrum of compound A in Example 1 are shown; the yellow line is the absorption spectrum and the blue line is the fluorescence emission spectrum.

[0034] Figure 3 This is the glutathione response curve of Compound A in Example 1.

[0035] Figure 4 This is a diagram showing the in vitro therapeutic effect of Compound A in Example 1 on PC3-PSMA+ cells.

[0036] Figure 5 This is a comparison of cell morphology before and after the treatment of PC3-PSMA+ cells with Compound A in Example 1.

[0037] Figure 6 This is a diagram showing the application of Compound A in Example 1 for cell imaging.

[0038] Figure 7 The graphs show that Compound A of Example 1 was used for in vivo tumor imaging at different times.

[0039] Figure 8This is a graph showing the tumor growth curve after injection of Compound A from Example 1 via the tail vein.

[0040] Figure 9 This is an H&E staining image of the main tissue and organ sections after the injection of Compound A in Example 1 via the tail vein.

[0041] Figure 10 This is a graph showing the main blood indices after injection of Compound A from Example 1 via the tail vein. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The test methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0043] The trident-shaped small molecule drug conjugate of the present invention is prepared by the following specific process:

[0044] S1. Weigh the catalyst, linker, and cytotoxic molecule in a molar ratio of (1-10):(1.05-2):1, add them into a container, then add the solvent, and stir for ≥6 hours to obtain a cytotoxic molecule-linker conjugate.

[0045] S2. Weigh the catalyst, spacer and cytotoxic molecule-linker conjugate in a molar ratio of (1-10):(1.05-2):1, add them to a container, then add the solvent, and stir for ≥6 hours to obtain a cytotoxic molecule-linker-spacer conjugate.

[0046] The spacer is selected from tert-butyl-2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (tBu-DUPA) or tert-butyl 4-aminobutyrate, tBu-DUPA corresponds to tert-Butyl 4-aminobutyrate corresponds to

[0047] S2. Weigh the catalyst, polyethylene glycol, and cytotoxic molecule-linker-spacer conjugate in a molar ratio of (1-10):(3.6-4):1, add them into a container, then add the solvent, and stir for ≥6 hours to obtain a trident structure.

[0048] S3. Weigh the catalyst, targeting ligand, and trident structure in a molar ratio of (1-10):(3.6-4):1, add them into a container, then add the solvent, and stir for ≥6 hours to obtain a trident small molecule drug conjugate.

[0049] The catalyst is selected from one or more of triethylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 1-ethyl-(3-dimethylaminopropyl)carbodiimide-1-hydroxybenzotriazole (HoBt), 1,3-dicyclohexylcarbodiimide (DCC), 1,3-dicyclohexylcarbodiimide-4-dimethylaminopyridine (DMAP), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0050] The solvent is selected from one or more of chloroform, dichloromethane (DCM), tetrahydrofuran, acetonitrile, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF).

[0051] The application of the trident-shaped small molecule conjugate drug of the present invention comprises the following steps:

[0052] (1) The trident-shaped small molecule drug conjugate is incubated with cells, and then the cells are imaged using a fluorescence microscope.

[0053] (2) The trident-shaped small molecule drug conjugate is injected into tumor-bearing mammals for fluorescence imaging.

[0054] (3) Incubate the trident-shaped small molecule drug conjugate with cells and observe the cell killing effect.

[0055] (4) Inject the trident-shaped small molecule drug conjugate into a mammalian disease model and then observe the therapeutic effect.

[0056] (5) The trident-shaped small molecule drug conjugate is injected into a mammalian disease model, and then blood indicators are measured.

[0057] The following are specific examples, which are intended to further illustrate the present invention. These examples are merely exemplary and should not be construed as limiting the scope of this application.

[0058] Example 1

[0059] The preparation method of trident-shaped small molecule drug conjugate compound A comprises the following steps:

[0060] S1. Dissolve p-nitrophenyl chloroformate (0.1 mmol) and bis(2-hydroxyethyl) disulfide (0.12 mmol) in DMF (3 mL), add triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a disulfide linker.

[0061] S2. Dissolve the disulfide linker (0.1 mmol) and the cytotoxic molecule 7-ethyl-10-hydroxycamptothecin (SN38, 0.12 mmol) in DMF (3 mL), add the catalyst triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a cytotoxic molecule-linker conjugate, which is recorded as SN38-SS molecule.

[0062] S3. Dissolve the SN38-SS molecule (0.1 mmol), spacer tBu-DUPA (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) in DCM (3 mL). Then add pyridine (48 μL) dropwise. Stir at room temperature for 6 h and add trifluoroacetic acid (1 mL). Pyridine and trifluoroacetic acid remove the tBu on tBu-DUPA. React at room temperature for 2 h, dry by rotary evaporation, and purify to obtain a cytotoxic molecule-linker-spacer conjugate, recorded as SN38-SS-DUPA.

[0063] S4, SN38-SS-DUPA (0.1 mmol), amino 24-tetra-polyethylene glycol valine tert-butyl ester (H2N-PEG 24 -CH2CH2COOtBu) (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as SN38-SS-3PEG. 24 .

[0064] S5, SN38-SS-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain compound A, which was recorded as SPP.

[0065] Example 2

[0066] The preparation method of the trident-shaped small molecule drug conjugate compound B is the same as the preparation steps of Example 1, except that tBu-DUPA in step S5 is replaced with fibroblast activation protein inhibitor (FAPI), comprising the following steps:

[0067] SN38-SS-3PEG 24 (0.1 mmol), targeting ligand FAPI (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound B.

[0068] Example 3

[0069] The preparation method of trident-shaped small molecule drug conjugate compound C is the same as the preparation steps in Example 1, except that tBu-DUPA in step S5 is replaced by folic acid, comprising the following steps:

[0070] SN38-SS-3PEG 24 (0.1 mmol), targeting ligand folic acid (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound C.

[0071] Example 4

[0072] The preparation method of trident-shaped small molecule drug conjugate compound D is the same as the preparation steps of Example 1, except that tBu-DUPA in step S5 is replaced by RGD polypeptide, comprising the following steps:

[0073] SN38-SS-3PEG 24 (0.1 mmol), targeting ligand RGD peptide (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound D.

[0074] Example 5

[0075] The preparation method of trident-shaped small molecule drug conjugate compound E is the same as the preparation steps of Example 1, except that tBu-DUPA in step S5 is replaced by Bombesin polypeptide, comprising the following steps:

[0076] SN38-SS-3PEG 24(0.1 mmol), targeting ligand Bombesin peptide (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound E.

[0077] Example 6

[0078] The preparation method of trident-shaped small molecule drug conjugate compound F is the same as the preparation steps of Example 1, except that tBu-DUPA in step S5 is replaced by octreotide peptide, comprising the following steps:

[0079] SN38-SS-3PEG 24 (0.1 mmol), targeting ligand Octreotide peptide (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound F.

[0080] Example 7

[0081] The preparation method of trident-shaped small molecule drug conjugate compound G comprises the following steps:

[0082] S1. Dissolve the linker Val-Cit-PAB (valine-citrulline, 0.1 mmol), the cytotoxic molecule 7-ethyl-10-hydroxycamptothecin (SN38, 0.12 mmol), the catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) in DMF (3 mL). Add pyridine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a cytotoxic molecule-linker conjugate, designated as SN38-Vc.

[0083] S2. SN38-Vc (0.1 mmol), spacer tBu-DUPA (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as SN38-Vc-DUPA.

[0084] S3, SN38-Vc-DUPA (0.1 mmol), H2N-PEG 24 -CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was designated as SN38-Vc-3PEG. 24 .

[0085] S4, SN38-Vc-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound G.

[0086] Example 8

[0087] The preparation method of trident-shaped small molecule drug conjugate compound H comprises the following steps:

[0088] S1. Dissolve the linker Val-Ala (valine-alanine, 0.1 mmol), the cytotoxic molecule 7-ethyl-10-hydroxycamptothecin (SN38, 0.12 mmol), the catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) in DMF (3 mL). Add pyridine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a cytotoxic molecule-linker conjugate, designated as SN38-Va.

[0089] S2. SN38-Va (0.1 mmol), spacer tBu-DUPA (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as SN38-Va-DUPA.

[0090] S3. Combine SN38-Va-DUPA (0.1mmol) and H2N-PEG 24-CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as SN38-Va-3PEG. 24 .

[0091] S4, SN38-Va-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound H.

[0092] Example 9

[0093] The preparation method of trident-shaped small molecule drug conjugate compound I comprises the following steps:

[0094] S1. The linker Phe-Lys (phenylalanine-lysine, 0.1 mmol), the cytotoxic molecule 7-ethyl-10-hydroxycamptothecin (SN38, 0.12 mmol), the catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DMF (3 mL). Pyridine (48 μL) was added dropwise and stirred at room temperature for 6 h. The mixture was purified to obtain a cytotoxic molecule-linker conjugate, which was designated as SN38-Pl.

[0095] S2. SN38-Pl (0.1 mmol), spacer tBu-DUPA (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as SN38-Pl-DUPA.

[0096] S3. Combine SN38-Pl-DUPA (0.1mmol) and H2N-PEG 24-CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as SN38-Pl-3PEG. 24 .

[0097] S4, SN38-Pl-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound I.

[0098] Example 10

[0099] The preparation method of trident-shaped small molecule drug conjugate compound J comprises the following steps:

[0100] S1. [(S)-1-[[(S)-1-[[4-(Hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamic acid (9H-fluoren-9-yl)methyl ester-methyl auristatin E (Fmoc-Val-Cit-PAB-MMAE, a conjugate of a cytotoxic molecule and a linker) (0.1 mmol), a spacer tBu-DUPA (0.12 mmol), a catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and the mixture was reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as MMAE-Vc-DUPA.

[0101] S2. Combine MMAE-Vc-DUPA (0.1mmol) and H2N-PEG 24 -CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as MMAE-Vc-3PEG. 24 .

[0102] S3, MMAE-Vc-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound J.

[0103] Example 11

[0104] The preparation method of trident-shaped small molecule drug conjugate compound K comprises the following steps:

[0105] S1. Dissolve p-nitrophenyl chloroformate (0.1 mmol) and bis(2-hydroxyethyl) disulfide (0.12 mmol) in DMF (3 mL), add triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a disulfide linker.

[0106] S2. Dissolve the disulfide linker (0.1 mmol) and the cytotoxic molecule doxorubicin hydrochloride (Dox, 0.12 mmol) in DMF (3 mL), add the catalyst triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a cytotoxic molecule-linker conjugate, which is recorded as Dox-SS.

[0107] S3. Dox-SS (0.1 mmol), spacer tBu-DUPA (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as Dox-SS-DUPA.

[0108] S4. Combine Dox-SS-DUPA (0.1mmol) and H2N-PEG 24 -CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as Dox-SS-3PEG. 24 .

[0109] S5, Dox-SS-3PEG 24 (0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound K.

[0110] Example 12

[0111] The preparation method of trident-shaped small molecule drug conjugate compound L comprises the following steps:

[0112] S1. Dissolve p-nitrophenyl chloroformate (0.1 mmol) and bis(2-hydroxyethyl) disulfide (0.12 mmol) in DMF (3 mL), add triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a disulfide linker.

[0113] S2. Dissolve the disulfide linker (0.1 mmol) and the cytotoxic molecule paclitaxel (Pac, 0.12 mmol) in DMF (3 mL), add the catalyst triethylamine (48 μL) dropwise, stir at room temperature for 6 h, and purify to obtain a cytotoxic molecule-linker conjugate, which is recorded as Pac-SS.

[0114] S3. Pac-SS (0.1 mmol), spacer tert-butyl 4-aminobutyrate (0.12 mmol), catalyst HoBt (0.12 mmol) and EDC·HCl (0.5 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain a cytotoxic molecule-linker-spacer conjugate, which was recorded as Pac-SS-DUPA.

[0115] S4. Combine Pac-SS-DUPA (0.1mmol) and H2N-PEG 24 -CH2CH2COOtBu (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was dried by rotary evaporation and purified to obtain a trident structure, which was recorded as Pac-SS-3PEG. 24 .

[0116] S5, Pac-SS-3PEG 24(0.1 mmol), targeting ligand tBu-DUPA (0.36 mmol), catalyst HoBt (0.36 mmol) and EDC·HCl (0.15 mmol) were dissolved in DCM (3 mL), and then pyridine (48 μL) was added dropwise. After stirring at room temperature for 6 h, trifluoroacetic acid (1 mL) was added and reacted at room temperature for 2 h. The mixture was rotary evaporated to dryness and purified to obtain compound L.

[0117] The characterization of compound A is shown in Figure 1 High-resolution mass spectra of Figure 1 The results show that compound A was successfully prepared. The absorption spectrum and fluorescence emission spectrum of compound A are shown in Figure 2 .from Figure 2 It can be seen that compound A has maximum absorption at 365nm and maximum emission at around 430nm. The absorption and emission in this band enable it to be used for cell fluorescence imaging.

[0118] Figure 3 The results showed that compound A could effectively break the disulfide bond linker and release the cytotoxic molecule SN38 under the action of glutathione at a concentration equivalent to the level in the tumor microenvironment (~5mM), verifying in vitro the potential of compound A to effectively release cytotoxic molecules in the tumor microenvironment to exert its killing effect.

[0119] Compound A was used for in vitro cell killing: Compound A was prepared into physiological isotonic PBS buffer solution and added to 96-well plates cultured with PC3-PSMA+ prostate cancer cells at different concentrations. After incubation for 72 hours, the cells were characterized using a standard MTT test. Figure 4 As shown, the cells in the control group (control) all had good cell viability, but the cells incubated with compound A showed a significant growth inhibition effect, indicating that the prepared compound A has a significant in vitro cancer cell killing ability. In addition, Figure 4 The results showed that compound A could effectively kill PC3-PSMA+ prostate cancer cells and exhibited concentration-dependent cytotoxicity.

[0120] Figure 5 The cell-killing ability of SPP was verified from cell morphology. Compared with untreated cells, cells treated with SPP for 24 hours showed obvious shrinkage and necrosis.

[0121] Compound A was used for cell imaging: Compound A (1 μmol) was incubated with PC3-PSMA+ prostate cancer cells for 15 min. The control group was incubated with Glu-urea-lys that can bind to prostate-specific membrane antigen in advance. The effect of compound A on cell imaging was then observed using a fluorescence microscope. The results are shown in Figure 2. Figure 6As shown, it can be seen that the fluorescence intensity of the control group incubated with Glu-urea-lys in advance was significantly reduced, indicating that compound A entered the cells through specific binding of antigen and antibody, verifying its in vitro targeting.

[0122] To further evaluate the targeting ability of SPP to prostate cancer cells in vivo, we selected the fluorescent molecule pyropheophytin A (PHA) instead of SN38 for in vivo fluorescence imaging. Compound A was injected into mice bearing PC3-PSMA+ subcutaneous tumors via the tail vein (20 nmol, 200 μL). Non-invasive imaging was performed using a small animal in vivo fluorescence imaging system 0.5 h, 1 h, 2 h, and 4 h later to observe the in vivo tumor accumulation process of compound A. Figure 7 After tail vein injection, compound A was gradually enriched in tumor tissue through active targeting.

[0123] Compound A was used for in vivo tumor inhibition therapy: Compound A was injected into mice bearing PC3-PSMA+ subcutaneous tumor models via the tail vein. The subcutaneous tumor size was measured after 0, 2, 4, 6, 8, 10, 12, and 14 days. The in vivo tumor inhibition effect of Compound A was confirmed by comparing the tumor growth curve with that of the control group (PBS). Figure 8 , the tumor growth curve after compound A was injected through the tail vein, and it was seen that compound A could effectively inhibit tumor growth.

[0124] To evaluate the biosafety of SPP, various organs were collected for H&E staining. Figure 9 The results showed that no lesions or inflammation were found in the sections of heart, liver, spleen, lung and kidney.

[0125] Blood was collected 24 hours after intravenous injection of SPP for routine blood examination to measure common blood parameters, including red blood cell (RBC), white blood cell (WBC) and platelet (PLT) counts. Figure 10 The results showed that all values ​​remained within the normal physiological range. At the same time, the common serum biochemical indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN) were all within the normal range, indicating normal liver and kidney function.

[0126] Example 13

[0127] The preparation steps are the same as those in Example 1, except that the monodisperse polyethylene glycol 24 in Example 1 is replaced by monodisperse polyethylene glycol 6.

[0128] Example 14

[0129] The preparation steps are the same as those in Example 1, except that the monodisperse polyethylene glycol 24 in Example 1 is replaced by monodisperse polyethylene glycol 12.

[0130] Example 15

[0131] The preparation steps are the same as those in Example 1, except that the monodisperse polyethylene glycol 24 in Example 1 is replaced by polydisperse polyethylene glycol 2000.

[0132] Example 16

[0133] The preparation steps are the same as those in Example 1, except that the monodisperse polyethylene glycol 24 in Example 1 is replaced by polydisperse polyethylene glycol 5000.

[0134] Example 17

[0135] The preparation steps are the same as those in Example 2, except that the monodisperse polyethylene glycol 24 in Example 2 is replaced by monodisperse polyethylene glycol 6.

[0136] Example 18

[0137] The preparation steps are the same as those in Example 2, except that the monodisperse polyethylene glycol 24 in Example 2 is replaced by monodisperse polyethylene glycol 12.

[0138] Example 19

[0139] The preparation steps are the same as those in Example 2, except that the monodisperse polyethylene glycol 24 in Example 2 is replaced by polydisperse polyethylene glycol 2000.

[0140] Example 20

[0141] The preparation steps are the same as those in Example 2, except that the monodisperse polyethylene glycol 24 in Example 2 is replaced by polydisperse polyethylene glycol 5000.

[0142] Example 21

[0143] The preparation steps are the same as those in Example 3, except that the monodisperse polyethylene glycol 24 in Example 3 is replaced by monodisperse polyethylene glycol 6.

[0144] Example 22

[0145] The preparation steps are the same as those in Example 3, except that the monodisperse polyethylene glycol 24 in Example 3 is replaced by monodisperse polyethylene glycol 12.

[0146] Example 23

[0147] The preparation steps are the same as those in Example 3, except that the monodisperse polyethylene glycol 24 in Example 3 is replaced by polydisperse polyethylene glycol 2000.

[0148] Example 24

[0149] The preparation steps are the same as those in Example 3, except that the monodisperse polyethylene glycol 24 in Example 3 is replaced by polydisperse polyethylene glycol 5000.

[0150] Example 25

[0151] The preparation steps are the same as those in Example 4, except that the monodisperse polyethylene glycol 24 in Example 4 is replaced by monodisperse polyethylene glycol 6.

[0152] Example 26

[0153] The preparation steps are the same as those in Example 4, except that the monodisperse polyethylene glycol 24 in Example 4 is replaced by monodisperse polyethylene glycol 12.

[0154] Example 27

[0155] The preparation steps are the same as those in Example 4, except that the monodisperse polyethylene glycol 24 in Example 4 is replaced by polydisperse polyethylene glycol 2000.

[0156] Example 28

[0157] The preparation steps are the same as those in Example 4, except that the monodisperse polyethylene glycol 24 in Example 4 is replaced by polydisperse polyethylene glycol 5000.

[0158] Example 29

[0159] The preparation steps are the same as those in Example 5, except that the monodisperse polyethylene glycol 24 in Example 5 is replaced by monodisperse polyethylene glycol 6.

[0160] Example 30

[0161] The preparation steps are the same as those in Example 5, except that the monodisperse polyethylene glycol 24 in Example 5 is replaced by monodisperse polyethylene glycol 12.

[0162] Example 31

[0163] The preparation steps are the same as those in Example 5, except that the monodisperse polyethylene glycol 24 in Example 5 is replaced by polydisperse polyethylene glycol 2000.

[0164] Example 32

[0165] The preparation steps are the same as those in Example 5, except that the monodisperse polyethylene glycol 24 in Example 5 is replaced by polydisperse polyethylene glycol 5000.

[0166] Example 33

[0167] The preparation steps are the same as those in Example 6, except that the monodisperse polyethylene glycol 24 in Example 6 is replaced by monodisperse polyethylene glycol 6.

[0168] Example 34

[0169] The preparation steps are the same as those in Example 6, except that the monodisperse polyethylene glycol 24 in Example 6 is replaced by monodisperse polyethylene glycol 12.

[0170] Example 35

[0171] The preparation steps are the same as those in Example 6, except that the monodisperse polyethylene glycol 24 in Example 6 is replaced by polydisperse polyethylene glycol 2000.

[0172] Example 36

[0173] The preparation steps are the same as those in Example 6, except that the monodisperse polyethylene glycol 24 in Example 6 is replaced by polydisperse polyethylene glycol 5000.

[0174] Example 37

[0175] The preparation steps are the same as those in Example 7, except that the monodisperse polyethylene glycol 24 in Example 7 is replaced by monodisperse polyethylene glycol 6.

[0176] Example 38

[0177] The preparation steps are the same as those in Example 7, except that the monodisperse polyethylene glycol 24 in Example 7 is replaced by monodisperse polyethylene glycol 12.

[0178] Example 39

[0179] The preparation steps are the same as those in Example 7, except that the monodisperse polyethylene glycol 24 in Example 7 is replaced by polydisperse polyethylene glycol 2000.

[0180] Example 40

[0181] The preparation steps are the same as those in Example 7, except that the monodisperse polyethylene glycol 24 in Example 7 is replaced by polydisperse polyethylene glycol 5000.

[0182] Example 41

[0183] The preparation steps are the same as those in Example 8, except that the monodisperse polyethylene glycol 24 in Example 8 is replaced by monodisperse polyethylene glycol 6.

[0184] Example 42

[0185] The preparation steps are the same as those in Example 8, except that the monodisperse polyethylene glycol 24 in Example 8 is replaced by monodisperse polyethylene glycol 12.

[0186] Example 43

[0187] The preparation steps are the same as those in Example 8, except that the monodisperse polyethylene glycol 24 in Example 8 is replaced by polydisperse polyethylene glycol 2000.

[0188] Example 44

[0189] The preparation steps are the same as those in Example 8, except that the monodisperse polyethylene glycol 24 in Example 8 is replaced by polydisperse polyethylene glycol 5000.

[0190] Example 45

[0191] The preparation steps are the same as those in Example 9, except that the monodisperse polyethylene glycol 24 in Example 9 is replaced by monodisperse polyethylene glycol 6.

[0192] Example 46

[0193] The preparation steps are the same as those in Example 9, except that the monodisperse polyethylene glycol 24 in Example 9 is replaced by monodisperse polyethylene glycol 12.

[0194] Example 47

[0195] The preparation steps are the same as those in Example 9, except that the monodisperse polyethylene glycol 24 in Example 9 is replaced by polydisperse polyethylene glycol 2000.

[0196] Example 48

[0197] The preparation steps are the same as those in Example 9, except that the monodisperse polyethylene glycol 24 in Example 9 is replaced by polydisperse polyethylene glycol 5000.

[0198] Example 49

[0199] The preparation steps are the same as those in Example 10, except that the monodisperse polyethylene glycol 24 in Example 10 is replaced by monodisperse polyethylene glycol 6.

[0200] Example 50

[0201] The preparation steps are the same as those in Example 10, except that the monodisperse polyethylene glycol 24 in Example 10 is replaced by monodisperse polyethylene glycol 12.

[0202] Example 51

[0203] The preparation steps are the same as those in Example 10, except that the monodisperse polyethylene glycol 24 in Example 10 is replaced by polydisperse polyethylene glycol 2000.

[0204] Example 52

[0205] The preparation steps are the same as those in Example 10, except that the monodisperse polyethylene glycol 24 in Example 10 is replaced by polydisperse polyethylene glycol 5000.

[0206] Example 53

[0207] The preparation steps are the same as those in Example 11, except that the monodisperse polyethylene glycol 24 in Example 11 is replaced by monodisperse polyethylene glycol 6.

[0208] Example 54

[0209] The preparation steps are the same as those in Example 11, except that the monodisperse polyethylene glycol 24 in Example 11 is replaced by monodisperse polyethylene glycol 12.

[0210] Example 55

[0211] The preparation steps are the same as those in Example 11, except that the monodisperse polyethylene glycol 24 in Example 11 is replaced by polydisperse polyethylene glycol 2000.

[0212] Example 56

[0213] The preparation steps are the same as those in Example 11, except that the monodisperse polyethylene glycol 24 in Example 11 is replaced by polydisperse polyethylene glycol 5000.

[0214] Example 57

[0215] The preparation steps are the same as those in Example 12, except that the monodisperse polyethylene glycol 24 in Example 12 is replaced by monodisperse polyethylene glycol 6.

[0216] Example 58

[0217] The preparation steps are the same as those in Example 12, except that the monodisperse polyethylene glycol 24 in Example 12 is replaced by monodisperse polyethylene glycol 12.

[0218] Example 59

[0219] The preparation steps are the same as those in Example 12, except that the monodisperse polyethylene glycol 24 in Example 12 is replaced by polydisperse polyethylene glycol 2000.

[0220] Example 60

[0221] The preparation steps are the same as those in Example 12, except that the monodisperse polyethylene glycol 24 in Example 12 is replaced by polydisperse polyethylene glycol 5000.

[0222] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and their scope of protection is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art on the basis of the present invention are all within the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims.

Claims

1. A trident-shaped small molecule drug conjugate, characterized in that: The trident-shaped small molecule drug conjugate is composed of a cytotoxic molecule, a linker, a spacer, and three targeting ligands. The cytotoxic molecule is coupled to the linker, and the targeting ligand is coupled to a polyethylene glycol chain. The polyethylene glycol chain and the linker are coupled together through the spacer. The structural formula of the trident-shaped small molecule drug conjugate is as follows: Where n is 6 to 13640, represents polyethylene glycol chains of varying lengths; D is a cytotoxic molecule, L is a linker, T is a targeting ligand, and the spacer is 2. A trident-shaped small molecule drug conjugate according to claim 1, characterized in that: The cytotoxic molecule is selected from one of doxorubicin hydrochloride, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, auristatin, auristatin derivatives, porphyrin, and porphyrin derivatives.

3. A trident-shaped small molecule drug conjugate according to claim 1, characterized in that: The linker is selected from a peptide linker or a disulfide linker.

4. A trident-shaped small molecule drug conjugate according to claim 3, characterized in that: The peptide linker is selected from one of valine-citrulline, phenylalanine-lysine, and valine-alanine.

5. The trident-shaped small molecule drug conjugate according to claim 1, characterized in that: The polyethylene glycol chains of different lengths are selected from one or more monodisperse polyethylene glycols with 6 to 48 chain segments, or one or more polydisperse polyethylene glycols with a molecular weight of 500 to 600,000.

6. A trident-shaped small molecule drug conjugate according to claim 1, characterized in that: The targeting ligand is one or more of a prostate cancer-specific membrane surface antigen targeting molecule, folic acid, bombesin polypeptide, octreotide polypeptide, RGD polypeptide, bombesin polypeptide, octreotide, melanocortin-1 receptor targeting polypeptide, and galactose targeting molecule.

7. A trident-shaped small molecule drug conjugate according to claim 6, characterized in that: The prostate cancer-specific membrane surface antigen targeting molecule is selected from tert-butyl-2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid.

8. The trident-shaped small molecule drug conjugate according to claim 1, characterized in that: The structural formula of the trident-shaped small molecule drug conjugate is:

9. A method for preparing the trident-shaped small molecule drug conjugate according to any one of claims 1 to 8, characterized in that: The steps include: connecting the cytotoxic molecule and the linker through an amidation reaction to generate a cytotoxic molecule-linker conjugate; linking the cytotoxic molecule-linker conjugate to the spacer through an amidation reaction to form a cytotoxic molecule-linker-spacer conjugate; The cytotoxic molecule-linker-spacer conjugate is connected to three polyethylene glycols through an amidation reaction to form a trident-like structure; The trifurcated structure is connected to three targeting ligands through an amidation reaction to generate a trifurcated small molecule drug conjugate.

10. Use of the trident-shaped small molecule drug conjugate according to claim 1 in the preparation of cancer therapeutic drugs.

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