A small-molecule conjugate compound targeting asgpr and a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and specifically discloses a small-molecule coupling compound targeting ASGPR, which has the following general formula: wherein X is a small-molecule ligand with the ability to target ASGPR; and Y is a derivative of cucurbitacin B. . The small-molecule coupling compound can be used in the preparation of a drug targeting ASGPR and having the functions of precise chemotherapy and radiotherapy sensitization. The small-molecule coupling compound prepared by the present application can achieve precise killing of liver cancer cells; the water solubility of cucurbitacin B is improved after coupling; the small-molecule coupling compound integrates precise chemotherapy and radiotherapy sensitization, and can significantly enhance the cancer-killing effect in combination with low-dose radiotherapy; and the small-molecule coupling compound has no obvious systemic toxicity and is well tolerated by patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a small molecule conjugate targeting ASGPR, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality rates worldwide. Most cases are closely related to chronic viral hepatitis, aflatoxin exposure, alcoholic liver disease, and metabolic-associated steatohepatitis. Due to atypical early symptoms, over 80% of patients are diagnosed at an advanced stage, losing the opportunity for surgery. Current first-line treatments for advanced HCC include multi-kinase inhibitors (such as sorafenib and lenvatinib) and immunotherapy combinations, but their objective response rates are low, median overall survival remains limited to 12-19 months, and drug resistance is prevalent, including tumor microenvironment-mediated immune evasion and drug failure due to kinase mutations. Although chemotherapy and radiotherapy still have some role in advanced HCC, their application is limited by systemic toxicity, off-target effects, and damage to normal tissues. Therefore, there is an urgent need to develop novel treatment strategies with higher selectivity and lower toxicity.

[0003] In recent years, active ingredients of traditional Chinese medicine have shown potential value in anti-tumor research. Cucurbitacin B (CuB), the main active ingredient in cucurbitacin tablets, possesses various biological activities, including anti-tumor, anti-chemical carcinogenicity, hepatoprotection, and enhancement of the body's immunity. It can induce DNA damage and arrest the cell cycle at the G2 / M phase, suggesting its potential for radiosensitization. However, CuB's poor water solubility, non-selective toxicity, and unfavorable pharmacokinetic properties severely limit its clinical application.

[0004] Therefore, it is urgent to solve technical problems such as poor targeting of liver cancer, defects in the physicochemical properties of drugs, limited therapeutic functions, and significant toxic side effects, in order to meet the clinical treatment needs of advanced HCC. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule conjugate compound that can effectively target ASGPR and enhance the clinical efficacy of cucurbitacin B.

[0006] Another objective of this invention is to provide a specific method for preparing the aforementioned small molecule coupling compound targeting ASGPR.

[0007] Another objective of this invention is to provide the above-mentioned small molecule conjugates targeting ASGPR as drugs that target ASGPR and have chemotherapy and radiotherapy functions.

[0008] This invention provides a small molecule coupling compound targeting ASGPR, with the following general formula:

[0009] Wherein, X is a small molecule ligand with the ability to target ASGPR; Y is a derivative of cucurbitacin B.

[0010] Small molecule-drug conjugates (SMDCs) enhance tumor-specific delivery efficiency by binding cytotoxic drugs to targeted ligands. Desialylate glycoprotein receptor (ASGPR), a high-affinity hepatocyte-specific receptor, can bind to galactose (Gal) or... N α-Acetylgalactosamine (GalNAc) binding enables efficient internalization and lysosomal release, and has been successfully used for liver-targeted delivery of GalNAc-siRNA conjugates (such as Givlaari®). Furthermore, ASGPR is highly expressed in HCC cells, making it an ideal target. Previous studies, such as PK2 (doxorubicin-GalNAc small molecule conjugate), have preliminarily demonstrated the feasibility of the ASGPR targeting strategy.

[0011] Based on this, this technical solution uses a small glycoside ligand that specifically binds to ASGPR as a targeted therapeutic carrier, and cucurbitacin B (CuB), which has multiple biological activities such as anti-tumor, anti-chemical carcinogenesis, liver protection, and enhancement of the body's immunity, and has significant anti-HCC activity, as the therapeutic drug. The structure of both is modified to ultimately achieve a connection between them using an ester-bonded structure. The two components are linked to form a small molecule conjugate targeting ASGPR. Because the conjugate contains an ester bond, after targeting ASGPR via a small carbohydrate ligand, it is cleaved by an esterase, releasing the drug CuB. This small molecule conjugate enhances the drug's targeting of HCC cells, improves its pharmacokinetic properties, and reduces systemic toxicity. Furthermore, by utilizing CuB's dual chemotherapeutic and radiosensitizing properties, it provides a new approach for the precise combination of chemotherapy and radiotherapy in HCC treatment.

[0012] The small molecule ligand with ASGPR targeting capability is a carbohydrate small molecule ligand, and the carbohydrate small molecule in the carbohydrate small molecule ligand is... N - Acetylgalactose, lactose, lactoside N - One of the acetylglucosamines; the small sugar molecule in the sugar molecule ligand is a trimer.

[0013] The structure of the cucurbitacin B derivative is as follows: .

[0014] The preparation method of the above-mentioned small molecule coupling compound targeting ASGPR includes the following steps: Step S1: Prepare cucurbitacin B derivatives and small molecule ligands with ASGPR targeting capabilities; The specific process for preparing cucurbitacin B derivatives is as follows: cucurbitacin B is used as the starting material and reacted with 6-azidohexanoic acid under EDCI and DMAP catalysis conditions. After esterification, compound I is obtained. The prepared small molecule ligand with ASGPR targeting ability is a carbohydrate small molecule ligand with an alkynyl group introduced at the end of the carbohydrate small molecule ligand; Step S2: Because the cucurbitacin B derivative has an azide group, and the terminal of the sugar molecule ligand has an alkynyl group, both react in CuSO4. . Under the catalytic conditions of 5H2O / sodium vitamin C, a copper-catalyzed click cyclization reaction occurs to yield the target compound.

[0015] The present invention also provides a medicament for the prevention, diagnosis and / or treatment of diseases related to ASGPR expression, comprising a pharmaceutical composition prepared by adding pharmaceutically acceptable, non-toxic and non-inert pharmaceutical carriers and / or excipients to the above-mentioned small molecule conjugates targeting ASGPR and their salts and hydrates as the main components.

[0016] The pharmaceutical carrier or excipient is one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients.

[0017] The pharmaceutical composition is prepared into various dosage forms using methods recognized in the pharmaceutical and food industries: sprays, aerosols, liquid preparations, or solid preparations; the liquid preparations include injections, suspensions, emulsions, solutions, or syrups; the solid preparations include tablets, capsules, granules, or powders.

[0018] The prevention, diagnosis, and / or treatment of diseases associated with ASGPR expression include liver cancer and tumors associated with ASGPR expression.

[0019] The drug can be administered orally, sublingually, or via mucosal dialysis; the injection can be administered intravenously, via intravenous drip, via intramuscular injection, via intraperitoneal injection, or via subcutaneous injection.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) In this invention, a small molecule of carbohydrate that can specifically bind to ASGPR, a molecular marker of liver cancer cells, is coupled with cucurbitacin B, the main active ingredient of cucurbitacin tablets, which has the function of "chemotherapy-radiotherapy", to obtain the target compound of this invention, namely a small molecule conjugate drug that targets ASGPR and has the function of "chemotherapy-radiotherapy". (2) Most of the compounds claimed in this invention exhibit good in vitro biological activity, among which the target small molecule conjugate IX has good selectivity and an IC50 of 1,500 against HepG2 and Huh-7 liver cancer cells. 50 The values ​​were 425 and 341 nM, respectively, and the IC50 values ​​for ASGPR-negative LX-2 cells were 425 and 341 nM, respectively. 50 Value greater than 5000 nM; (3) The present invention demonstrates through mechanism experiments that small molecule conjugate compound IX significantly induces the generation of ROS in liver cancer cells, causes DNA damage, and induces liver cancer cells to remain in the most radiosensitive G2 / M phase. When combined with low-dose radiotherapy (2Gy), it can increase the formation of γH2AX focal points, activate DNA damage repair, block cell processes, reduce the survival rate of clone formation, and achieve a radiosensitizing effect. (4) When the small molecule conjugate compound IX claimed in this invention is used in combination with low-dose radiotherapy, the combination of multiple small molecule conjugate compounds IX with 2 Gy radiotherapy shows better in vivo tumor control effect than single therapy, highlighting its application potential in precision radiotherapy and chemotherapy for liver cancer. Attached Figure Description

[0021] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a differential graph showing the expression of ASGPR protein using Western Blot in Example 6 of the present invention; Figure 2 The water solubility of the small molecule coupling compound IX, a trimeric sugar ligand in Example 8 of this invention; Figure 3 This is a graph showing the effect of different concentrations of the small molecule conjugate compound IX on the cell cycle of HepG2, Huh-7 and LX-2 cells in Example 10 of the present invention. Figure 4 This is a diagram showing the effect of the 5 µM small molecule conjugate compound IX in Example 10 of the present invention on the cell cycle of HepG2 and Huh-7 liver cancer cells at different treatment times. Figure 5 The images are crystal violet stained images taken on day 14 of HepG2 and Huh-7 liver cancer cells in Example 11 of this invention after treatment with different concentrations of small molecule conjugate compound IX and 2 Gy ionizing radiation. Figure 6 This is a fluorescence image of the focus of γH2AX on double-strand DNA breaks in HepG2 and Huh-7 liver cancer cells, induced by small molecule conjugate compound IX alone or in combination with low-dose radiotherapy in Example 12 of this invention. Figure 7This is a schematic diagram of the study in the mouse HepG2 liver cancer xenograft model in Example 14 of the present invention; Figure 8 This is a graph showing the mouse tumor growth curve, tumor inhibition rate, and mouse body weight change in Example 14 of the present invention. Figure 9 This is a staining image of mouse H&E tissue in Example 14 of the present invention; Figure 10 This is an immunohistochemical staining image of γH2AX in mouse tumors in Example 14 of the present invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Various substitutions and modifications can be made based on ordinary technical knowledge and conventional means in the art without departing from the above-described technical concept of the present invention, and all such substitutions and modifications should be included within the scope of the present invention.

[0023] To make the objectives, process conditions, and advantages of this invention clearer, the invention will be further described in detail with reference to the following embodiments. The specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0024] This invention provides a small molecule coupling compound targeting ASGPR, with the following general formula:

[0025] Wherein, X is a small molecule ligand with the ability to target ASGPR; Y is a derivative of cucurbitacin B.

[0026] In the embodiments, the trimer is used N Small carbohydrate molecules prepared from acetylgalactose serve as small molecule ligands capable of targeting ASGPR.

[0027] The trimer N The specific structures of the small carbohydrate molecules obtained from acetylgalactose preparation are as follows:

[0028] Trimer NThe preparation process of the acetylgalactose small molecule ligand is as follows: Using tromethamine as the initial raw material, it undergoes a Mac addition reaction with butyl acrylate under the action of NaOH to obtain compound II; compound II reacts with benzyl chloroformate under the action of Na2CO3 to obtain amino-protected compound III; compound III undergoes detert-butylation under the catalysis of trifluoroacetic acid to obtain compound IV; compound IV reacts with propynyl tetraglycolic acid under the catalysis of HBTU and HOBT, undergoing amide condensation to obtain compound V; compound V undergoes deCbz protecting group removal under the catalysis of 10% Pd / C and H2 to obtain compound VI; compound VI reacts with propynyl tetraglycolic acid under the catalysis of HBTU and HOBT, undergoing amidation to obtain compound VII; compound VII undergoes deacetylation under NaOMe / MeOH conditions to obtain the trimer. N -Acetogalactose small molecule ligand compound VIII; The structure of the cucurbitacin B derivative is as follows: .

[0029] The specific process for preparing the cucurbitacin B derivative (compound I) is as follows: cucurbitacin B is used as the starting material and reacted with 6-azidohexanoic acid under EDCI and DMAP catalysis conditions. After esterification, compound I is obtained.

[0030] Then, the prepared compound I is combined with the trimer N The target product is prepared by reacting with the acetylgalactose small molecule ligand (compound VIII). The specific process is as follows: Compound I and compound VIII underwent a copper-catalyzed click cyclization reaction under CuSO4·5H2O / sodium vitamin C catalysis, followed by purification by preparative HPLC to obtain purified small molecule coupling compound IX.

[0031] Example 1: This embodiment provides a specific preparation process for cucurbitacin B derivatives.

[0032] The synthetic route for cucurbitacin B derivative (i.e., compound I) is as follows:

[0033] The specific preparation process is as follows: Cucurbitacin B (34 mg, 0.05 mmol), DMAP (6 mg, 0.05 mmol), EDCl (19 mg, 0.1 mmol), and Boc-6-Ahx-OH (23 mg, 0.1 mmol) were dissolved in 5 mL of anhydrous 1,2-dichloroethane under stirring, and Et3N (28 µL, 0.2 mmol) was added. The mixture was stirred at 60 °C for 16 h, and the reaction progress was monitored by silica gel thin-layer chromatography (TLC). The reaction was quenched by adding saturated sodium bicarbonate aqueous solution and extracted with dichloromethane (15 mL × 3 times). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 2:1) to give compound I (54.6 mg, 60% yield) as a white solid.

[0034] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.13 (d, J = 15.6 Hz, 1H), 6.40 (d, J = 15.6Hz, 1H), 5.79 (d, J = 5.0 Hz, 1H), 5.48 (dd, J = 13.6, 5.4 Hz, 1H), 5.19 (t, J = 8.1 Hz, 1H), 4.62 (s, 1H), 4.29 (s, 1H), 3.28 (t, J = 7.0 Hz, 2H), 3.24(d, J = 15.7 Hz, 1H), 3.10 (dd, J = 12.2, 6.0 Hz, 2H), 2.80 (d, J = 12.1 Hz, 1H), 2.74 (d, J = 14.7 Hz, 1H), 2.69 (d, J= 7.5 Hz, 1H), 2.52 – 2.29 (m,4H), 2.14 – 2.07 (m, 4H), 2.05 – 1.84 (m, 9H), 1.75 – 1.60 (m, 11H), 1.58 (s,3H), 1.57 (s, 3H), 1.55 – 1.49 (m, 4H), 1.48 (d, J = 7.6 Hz, 5H), 1.43 (s,9H), 1.41 (s, 3H), 1.36 (s, 2H), 1.32 (d, J = 3.6 Hz, 6H), 1.30 – 1.27 (m,9H), 1.24 (d, J = 4.7 Hz, 8H), 1.09 (s, 3H), 1.02 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 211.95, 205.66, 200.88, 172.95, 172.59, 169.64, 152.67, 139.72, 120.52, 119.28, 79.15, 77.66, 77.24, 73.44, 73.22, 60.42, 54.09, 51.27, 51.23, 49.93, 48.61, 48.46, 47.96, 43.25, 42.10, 34.35, 33.75, 33.62, 31.94, 29.71, 28.80, 28.54, 28.44, 26.67, 26.43, 26.22, 26.15, 24.41, 24.24, 23.74,23.71, 22.70, 21.91, 21.30, 19.96, 19.74, 18.74, 14.21, 14.13. m / z ):811.3 [M-Boc+H] + . Example 2: This embodiment provides a trimer. N The specific preparation process and synthetic route for the acetylgalactose small molecule ligand (i.e., compound X) are as follows:

[0035] (1) The specific preparation process of compound II is as follows: To a solution of 6.1 g (50 mmol) 2-amino-2-(hydroxymethyl)-1,3-propanediol in 50 mL of dimethyl sulfoxide under stirring, 5 mL of 40% (w / v) sodium hydroxide aqueous solution was added at 0 °C. Tert-butyl acrylate (64 g, 500 mmol) dissolved in 20 mL of dimethyl sulfoxide was slowly added dropwise to the reaction system, and the reaction mixture was stirred overnight at room temperature. Water (100 mL) was added to the reaction solution, followed by extraction with ethyl acetate (50 mL × 3 times). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate 3:1) to give compound II (18.9 g, 75% yield), a colorless oily liquid.

[0036] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, CDCl3) δ 3.62 (t, J = 6.4 Hz, 6H), 3.29 (s, 6H), 2.43(t, J = 6.4 Hz, 6H), 1.43 (s, 27H); 13 C NMR (101 MHz, CDCl3) δ 170.86, 80.34,72.84, 67.07, 55.88, 36.27, 28.05. MS-ESI ( m / z ): 506.2 [M+H] + . (2) The specific preparation process of compound III is as follows: Compound II (5.1 g, 10 mmol) was dissolved in a dichloromethane / water mixture (50 mL, 1:1 v / v), and sodium carbonate (10.1 g, 10 mmol) was added under vigorous stirring. The reaction mixture was placed in an ice-water bath, and then benzyl chloroformate (4.2 mL, 30 mmol) was slowly added dropwise. After reacting for 30 minutes, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL × 2 times). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate 10:1) to give compound III (5.2 g, 81% yield) as a colorless oily liquid.

[0037] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H), 5.30 (s, 1H), 5.03 (s,2H), 3.66 (s, 6H), 3.63 (t, J = 6.5 Hz, 6H), 2.43 (t, J = 6.4 Hz (6H), 1.43 (s, 27H); 13 C NMR (101 MHz, CDCl3) δ 170.81, 155.10, 136.71, 128.36, 127.94,127.85, 80.44, 69.34, 67.05, 66.07, 58.68, 36.19, 28.05. MS-ESI ( m / z ): 640.0[M+H] + . (3) The specific preparation process of compound IV is as follows: compound V (3.8 g, 6 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. The reaction solution was stirred at room temperature for 4 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was evaporated three times with toluene (5 mL) and dried under high vacuum to obtain the trifluoroacetate salt of compound IV. This compound does not require further purification and can be used directly in subsequent reactions. 1 H NMR (400 MHz, CDCl3) δ 10.96 (s, 3H), 7.34 – 7.29 (m, 5H), 5.04 (s, 2H), 3.76 – 3.53 (m, 12H), 2.56 (t, J = 6.1 Hz, 6H); 13 C NMR (101 MHz, CDCl3) δ 177.54, 159.55, 133.22, 128.85, 128.63,128.01, 69.47, 66.37, 53.40, 34.59. MS-ESI ( m / z ): 472.0 [M+H] + . (4) The specific preparation process of compound V is as follows: Propylene tetraglycolic acid (3.0 g, 6 mmol) and compound IV (471 mg, 1 mmol) were dissolved in anhydrous DMF (15 mL). Subsequently, HBTU (2.2 g, 6 mmol), HOBt (810 mg, 6 mmol), and DIEA (2.0 mL, 12 mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane (DCM, 100 mL) and washed with saturated sodium bicarbonate aqueous solution (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol 10:1) to give compound V (674 mg, yield 35%), a pale yellow oily liquid.

[0038] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H), 7.21 (t, J = 5.8 Hz, 3H), 6.92 (t, J = 5.7 Hz, 3H), 6.78 (d, J = 9.0 Hz, 2H), 5.76 (s, 1H), 5.35(dd, J = 8.9, 3.2 Hz, 3H), 5.18 (dd, J = 11.2, 3.4 Hz, 3H), 5.01 (s, 2H), 4.60 (d, J = 8.4 Hz, 3H), 4.27 – 3.99 (m, 9H), 3.97 – 3.85 (m, 6H), 3.68 –3.64 (m, 12H), 3.53 – 3.43 (m, 3H), 3.29 – 3.09 (m, 12H), 2.41 (t, J = 5.5Hz, 6H), 2.29 – 2.15 (m, 9H), 2.14 (s, 9H), 2.06 (d, J = 16.6 Hz, 6H), 2.04(s, 9H), 1.99 (s, 9H), 1.93 (s, 9H), 1.79 – 1.47 (m, 18H); 13 C NMR (101 MHz, CDCl3) δ173.80, 172.04, 171.02, 170.57, 170.50, 170.31, 155.68, 135.59,128.53, 128.20, 127.99, 101.09, 70.52, 70.28, 69.60, 69.38, 67.43, 66.68,61.46, 58.81, 51.03, 36.86, 36.07, 35.99, 35.89, 35.88, 29.56, 29.54, 28.27,23.28, 22.52, 20.69. MS-ESI ( m / z ): 964.6 [M+2H] 2+ . (5) The specific preparation process of compound VI is as follows: Compound V (578 mg, 0.3 mmol) was dissolved in methanol (10 mL), and 5 drops of acetic acid and 10% palladium on carbon (100 mg) were added. The reaction mixture was stirred at room temperature for 4 hours under a hydrogen atmosphere. After the reaction was complete, the reaction solution was filtered through a diatomaceous earth mat, and the filtrate was concentrated under reduced pressure to give crude product VI, a pale yellow oily liquid. This compound did not require further purification and was used directly in subsequent reactions.

[0039] MS-ESI ( m / z ): 897.2 [M+2H] 2+ . (6) The specific preparation process of compound VII is as follows: Alkyno-PEG5-acid (60 mg, 0.2 mmol) and compound VI (358 mg, 0.2 mmol) were dissolved in anhydrous DMF (8 mL). HBTU (113 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol), and DIEA (104 µL, 0.6 mmol) were then added to the solution, and the reaction mixture was stirred for 5 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with saturated brine (10 mL × 2 times). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (pre-HPLC) [Shimadzu LC-20AP system; column: Ultimate 5 μm XB-C18 Prep-HPLC Column (120 Å, 250 mm × 21.2 mm); flow rate: 8 mL / min; mobile phase A: 0.1% NH4HCO3 aqueous solution; mobile phase B: acetonitrile; gradient program: initial 10% B, hold for 10 min, increase to 50% B within 40 min and hold for 5 min, then increase to 90% B within 5 min and hold for another 5 min] to give compound VII (289 mg, yield 71%) as a pale yellow oily liquid.

[0040] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.29 (t, J = 6.0 Hz, 3H), 7.02 (t, J = 6.1Hz, 3H), 6.84 (s, 1H), 6.82 (s, 1H), 6.78 (s, 1H), 5.34 (d, J = 3.1 Hz, 3H), 5.18 (dd, J = 11.2, 3.3 Hz, 3H), 4.61 (d, J = 8.4 Hz, 3H), 4.18 (d, J= 2.2Hz, 3H), 4.17 – 4.01 (m, 9H), 3.96 – 3.87 (m, 6H), 3.70 – 3.65 (m, 21H), 3.63(s, 4H), 3.62 (s, 4H), 3.61 (s, 4H), 3.54 – 3.46 (m, 3H), 3.34 – 3.17 (m,12H), 2.47 (t, J = 2.2 Hz, 1H), 2.45 – 2.38 (m, 8H), 2.31 – 2.16 (m, 15H), 2.14 (s, 9H), 2.03 (s, 9H), 1.98 (s, 9H), 1.94 (s, 9H), 1.79 – 1.52 (m, 18H); 13 C NMR (101 MHz, CDCl3) δ 173.70, 172.09, 170.99, 170.49, 170.48, 170.32, 101.01, 79.54, 74.80, 70.50, 70.42, 70.36, 70.33, 70.28, 70.25, 70.11, 69.43, 69.31, 68.99, 67.43, 66.69, 61.46, 58.33, 51.02, 36.79, 36.12, 36.08, 36.00, 35.94, 35.92, 29.57, 28.28, 23.28, 22.48, 20.69. MS-ESI ( m / z ): 1040.2 [M+2H] 2+ 693.9 [M+3H] 3+ . (7) The specific preparation process of compound VIII is as follows: A 0.5 M sodium methoxide solution was added dropwise to an 8 mL stirred solution of compound VII (203 mg, 0.1 mmol) in methanol until the pH of the reaction system reached 9–10. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, formic acid was added to quench the reaction, and the mixture was concentrated under reduced pressure to give crude product VIII. This compound did not require further purification and was used directly in subsequent reactions.

[0041] MS-ESI ( m / z ): 851.2 [M+2H] 2+ 567.8 [M+3H] 3+ , 1723.8 [M+Na] + . Example 3: This embodiment is based on the above-mentioned polymer. N The acetylgalactose small molecule ligand was linked to compound I in the cucurbitacin B derivative to obtain three small molecule coupling compounds. The specific process is as follows: (3) The synthetic route of the small molecule coupling compound IX is as follows:

[0042] The specific preparation process of the small molecule coupling compound IX is as follows: Compound I (14 mg, 0.02 mmol) and compound VIII (34 mg, 0.02 mmol) were dissolved in a tetrahydrofuran / water mixture (5 mL, 1:1 v / v). Copper sulfate pentahydrate (5 mg, 0.02 mmol) and sodium ascorbate (8 mg, 0.04 mmol) were added sequentially to the stirred reaction mixture under an argon atmosphere. The reaction was carried out at room temperature for 5 hours. After the reaction was completed, the solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (pre-HPLC) [Shimadzu LC-20AP system; column: Ultimate 5 μm XB-C18 Prep-HPLC Column (120 Å, 250 mm × 21.2 mm); flow rate: 8 mL / min; mobile phase A: 0.1% NH4HCO3 aqueous solution; mobile phase B: acetonitrile; gradient program: initial 10% B, hold for 10 min, increase to 50% B within 40 min and hold for 5 min, then increase to 90% B within 5 min and hold for another 5 min] to obtain the trimer GalNAc small molecule conjugate IX (22 mg, yield 46%), as a white solid.

[0043] That 1 The H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.08 (s, 1H), 7.85 (s, 3H), 7.75 (s, 3H), 7.64 (d, J = 8.8 Hz, 3H), 7.14 (s, 1H), 6.80 (q, J = 16.1 Hz, 2H), 5.72 (s,1H), 5.65 (dd, J = 12.6, 4.4 Hz, 1H), 5.09 (s, 1H), 4.64 (d, J= 4.5 Hz, 3H), 4.57 (d, J = 5.8 Hz, 3H), 4.50 (d, J = 5.0 Hz, 7H), 4.32 (s, 2H), 4.21 (d, J = 8.2 Hz, 3H), 3.74 – 3.61 (m, 13H), 3.58 – 3.48 (m, 45H), 3.37 – 3.26 (m,9H), 3.13 (d, J = 11.8 Hz, 2H), 3.03 (s, 12H), 2.41 (d, J = 14.9 Hz, 1H),2.37 – 2.23 (m, 12H), 2.03 (t, J = 6.5 Hz, 6H), 1.97 (s, 3H), 1.82 (d, J =24.1 Hz, 15H), 1.73 – 1.61 (m, 2H), 1.61 – 1.37 (m, 27H), 1.25 (d, J = 15.8Hz, 17H), 1.16 (s, 3H), 0.90 (s, 3H), 0.72 (s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ213.55, 206.79, 204.72, 172.77, 172.26, 170.80, 170.26, 169.99, 149.18, 144.44, 140.33, 124.29, 122.21, 120.22, 101.91, 80.01, 79.36, 75.79, 73.84, 72.08, 70.31, 70.22, 70.02, 69.49, 68.82, 68.46, 68.11, 67.89, 67.39, 64.08, 61.06, 52.64, 51.33, 50.57, 49.70, 48.31, 47.89, 46.06, 46.04, 42.66, 37.14,36.95, 36.85, 36.59, 35.67, 33.52, 32.88, 32.36, 29.89, HRMS (ESI-Q-TOF): calcd for C 113 H 188 N 13 O 42 + [M + H] + , 2399.2969; found,2400.3069; [M + Na] + , 2421.2789; found, 2421.2849. Retention time: 14.029min, HPLC purity: 96.20%. Example 4: This embodiment describes the in vitro antitumor activity test (IC50) of different cell lines for the small molecule conjugate drug (i.e., compound IX) prepared above. 50 nM) experiment.

[0044] First, the levels of ASGPR protein expression in different cell lines were confirmed using Western blotting, immunofluorescence, and flow cytometry.

[0045] Western blot analysis was used to analyze the expression levels of ASGPR protein in HepG2, Huh-7, and LX-2 cells. The specific steps were as follows: Cells were lysed with RIPA lysis buffer (P0013B, Beyotime) to extract total protein. An equal volume of protein (50 μg) was separated by gel electrophoresis and transferred to a PVDF membrane. Primary antibodies used were rabbit anti-ASGPR antibody (11739-1-AP, Proteintech; 1:1000) and GAPDH antibody (81640-5-RR, Proteintech; 1:5000); secondary antibodies were HRP-labeled goat anti-rabbit antibody (RGAR001, Proteintech; 1:5000) and goat anti-mouse antibody (RGAM001, Proteintech), diluted 1:5000. After incubation and washing, enhanced chemiluminescence (ECL) was used for color development, and the results were analyzed using ChemiDoc. TM Images were acquired using the MP imaging system (Bio RAD, CA, USA). Experimental results are as follows: Figure 1 As shown, ASGPR is highly expressed in cells, lowly expressed in Huh-7 cells, and not expressed in LX-2 cells.

[0046] Next, the MTT assay was used to evaluate the in vitro antitumor activity of the conjugated drug (compound IX) prepared in this invention against HepG2, Huh-7, and LX-2 cells. Specific steps: Cells in logarithmic growth phase were seeded in 96-well plates (5 × 10³ cells per well, 200 µL of culture medium) and incubated overnight at 37°C and 5% CO2. After removing the culture medium, different concentrations of the compound solution were added, and incubation continued for 72 hours (37°C, 5% CO2). Subsequently, 20 µL of MTT solution (ST1537, Beotime; 5 mg / mL) was added to each well, and incubation continued for another 4 hours. The supernatant was discarded, and DMSO was added to dissolve the formazan crystals formed. The absorbance (OD value) was measured at 570 nm using a microplate reader, and concentration-response curves were plotted using GraphPad Prism 5.0 software (GraphPad Software). The half-maximal inhibitory concentration (IC50) of the compound on cells was calculated. 50 (nM). Finally, the experimental results are shown in Table 1.

[0047] Table 1. In vitro antiproliferative activity (IC50) of the compounds 50 , nM)

[0048] The results of in vitro activity tests are shown in Table 1. Compound X, a small molecule ligand of both the monomeric and trimeric sugars, did not show significant inhibitory effects on the proliferation of HepG2, Huh-7, or LX-2 cells, confirming its non-cytotoxic properties. In contrast, free CuB exhibited potent broad-spectrum cytotoxicity, validating its role as a highly efficient cytotoxic payload. The trimeric sugar conjugate IX showed excellent selectivity: it possessed strong anti-proliferative activity against HepG2 (425 ± 49 nM) and Huh-7 (341 ± 32 nM) cells, while exhibiting minimal toxicity to LX-2 cells (IC50). 50 > 5000 nM). This result indicates that the small molecule conjugate IX can specifically kill ASGPR-positive tumor cells and has a higher safety profile compared to free CuB.

[0049] Example 5: This embodiment also uses small molecule coupling compound IX as the research object to test its water solubility and plasma stability.

[0050] The water solubility and plasma stability of the small molecule conjugate IX were determined by HPLC. The solubility of CuB or compound IX in PBS (pH 7.4) was determined using centrifugation. The simplified procedure is as follows: An excess of the target compound was added to PBS in a 1.0 mL microcentrifuge tube and vortexed vigorously for 1 minute to form a supersaturated suspension. The suspension was then equilibrated for 24 hours at room temperature with continuous shaking (e.g., using a rotary mixer). Undissolved material was then separated by centrifugation at 13,000 rpm for 5 minutes at 25°C. The supernatant was carefully collected using a pipette, taking care to avoid disturbing the precipitate. Solubility was calculated by comparing the results with those obtained from standard solutions of CuB or compound IX in DMSO.

[0051] The stability test of compound IX in human plasma, rat plasma, and PBS (pH 7.4, negative control) was conducted as follows: Compound IX was dissolved in plasma or PBS preheated to 37°C to prepare an incubation system with a final concentration of 500 μM. The mixture was incubated in a 37°C water bath. Samples were taken at preset time points (0, 0.25, 0.5, 1, 2, 4, 8, 24, or 48 hours) and immediately mixed with ice-cold methanol to terminate the enzymatic reaction and precipitate plasma proteins. The quenched samples were then vortexed vigorously for 1 minute and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was carefully collected and immediately analyzed by HPLC.

[0052] Water solubility test results are as follows Figure 2As shown, compound IX exhibits a solubility exceeding 66,000 µM in PBS (pH 7.4), representing a more than 6,600-fold increase compared to unmodified CuB (solubility approximately 10 µM). This suggests that glycosylation coupling modification of the active ingredient cucurbitacin B can significantly enhance its water solubility and optimize its pharmacokinetic properties.

[0053] Example 6: This embodiment provides an experiment (flow cytometry) showing the effect of small molecule conjugate compound IX on the cell cycle of different cell lines.

[0054] The effect of different concentrations of small molecule conjugate IX on cell cycle was analyzed by flow cytometry. Specifically, HepG2, Huh-7, and LX-2 cells were incubated with DMSO and different concentrations of small molecule conjugate IX for 24 hours, respectively. After incubation, cells were fixed with pre-cooled methanol and incubated overnight at 4°C, followed by staining with PI / RNase (C1052, Beyotime). Flow cytometry analysis was performed using a BD LSRFortessa™ flow cytometer (BD Biosciences).

[0055] To elucidate the antitumor mechanism of the small molecule conjugate compound IX, we used flow cytometry to examine its effect on cell cycle progression. HepG2, Huh-7, and LX-2 cells were treated with different concentrations of SMDC 23 for 24 hours, followed by staining with propidium iodide (PI). Figure 3 As shown, the small molecule conjugate IX induced dose-dependent G2 / M phase arrest in ASGPR-positive HepG2 and Huh-7 cells. For example, treatment with 5, 10, and 20 µM of the small molecule conjugate IX increased the proportion of cells in the G2 / M phase in HepG2 cells from 6.40% in the untreated group to 24.10%, 33.12%, and 37.19%, respectively. This arrest was accompanied by a corresponding decrease in the proportion of cells in the G0 / G1 and S phases. In contrast, the small molecule conjugate IX had little effect on the cell cycle distribution of ASGPR-negative LX-2 cells, consistent with its selective cytotoxic characteristics. Further research revealed that the small molecule conjugate IX induced a time-dependent accumulation of G2 / M phase cells: after 48 hours of treatment with 5 µM of the small molecule conjugate IX, the proportion of G2 / M phase cells in HepG2 cells reached 25.90%, compared to 18.04% in the 12-hour treatment group, with a peak effect observed at 24 hours. Figure 4 As shown.

[0056] Example 7: This embodiment verifies the radiosensitizing effect of small molecule conjugate compound IX on HepG2 and Huh-7 liver cancer cells: Given that the small molecule conjugate IX specifically arrests hepatocellular carcinoma cells in the radiosensitive G2 / M phase, we will conduct a colony formation assay to investigate its radiosensitizing effect on HepG2 and Huh-7 hepatocellular carcinoma cells. Specifically, 1500 cells were seeded into each well of a 6-well plate, incubated overnight with the small molecule conjugate IX, and the cells were irradiated with 2 Gy after 24 hours. The culture medium was then replaced with fresh medium and the cells were cultured for another 14 days. After the experiment, the cells were fixed with methanol, stained with crystal violet, and colonies were counted. All experiments were repeated in duplicate. Furthermore, to evaluate the synergistic antitumor effect of the small molecule conjugate IX combined with low-dose irradiation (2 Gy) in HepG2 and Huh-7 cells, 1×10⁶ cells were added to the plate. 4 Cells were seeded per well in culture plates and pretreated with different concentrations (0, 2.5, 5, 10, 20 µM) of small molecule conjugate IX in complete culture medium for 24 hours. After drug removal, cells were irradiated with 2 Gy and cultured for another 24 hours. Cell viability was then assessed using the Beyo3D™ Calcein AM / PI assay kit (S1371S, Beyotime) strictly following the manufacturer's instructions. Cells were stained in the dark at 37°C for 30 minutes, followed by imaging analysis using a fluorescence microscope.

[0057] Based on the observed effects of small molecule conjugate IX on the cell cycle (i.e. Figure 4 We pretreated HepG2 and Huh-7 cells with different concentrations of the small molecule conjugate compound IX for 24 hours, followed by 2 Gy irradiation, to assess its effect on colony formation and survival. Crystal violet staining after 14 days showed... Figure 5 As shown, compared with the use of small molecule conjugate IX alone or irradiation alone, cells pretreated with small molecule conjugate IX before irradiation formed significantly smaller and fewer colonies. In summary, these findings demonstrate that small molecule conjugate IX can enhance the radiosensitivity of HCC cells and improve the therapeutic effect of radiotherapy when used in combination.

[0058] Example 8: This embodiment verifies the effects of small molecule conjugate compound IX on double-strand DNA breaks, γH2AX focal formation, and activation of DNA damage repair signaling pathways in liver cancer cells HepG2 and Huh-7.

[0059] Since radiotherapy can kill tumor cells by inducing DNA double-strand breaks (DSBs), and γH2AX is a significant molecular marker of DSBs, we will use immunofluorescence assays to detect the DNA damage response of HepG2 and Huh-7 cells in combination with low-dose radiotherapy using small molecule conjugate IX. Specifically, HCC cells (HepG2 and Huh-7) cultured in confocal glass-bottom dishes (BS-20-GJM, Biosharp Life Sciences) were treated with small molecule conjugate IX (0, 2.5, or 5 µM) for 24 hours, followed by 2 Gy radiotherapy. Samples were collected 2 hours post-irradiation. After washing with PBS, the cells were fixed with 100% cold methanol for 10 minutes. Non-specific binding was blocked with 5% bovine serum albumin (BSA) for 2 hours. Subsequently, cells were incubated overnight at 4 °C with anti-γH2AX antibody (ET1602-2, HUABIO; 1:500), followed by co-incubation with Cy3-labeled goat anti-rabbit secondary antibody (A0516, Beyotime; 1:500). Cell nuclei were counterstained with DAPI, and fluorescence images were subsequently acquired using an Andor Dragonfly 200 confocal microscope.

[0060] Simultaneously, Western blot analysis was used to investigate the effects of small molecule conjugate IX synergistically with low-dose radiotherapy on DNA damage and repair signaling pathways in HepG2 cells. Specifically, HepG2 cells were pretreated with small molecule conjugate IX and then irradiated with 2 Gy. Two hours after irradiation, cells were washed with PBS, scraped, and lysed using RIPA lysis buffer (P0013B, Beyotime) containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit (P0012S, Beyotime). Equal amounts of protein were separated by 10% SDS-PAGE and electroporated to a PVDF membrane. After blocking with TBST solution containing 5% BSA at room temperature for 1 hour, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-γH2AX (ET1602-2, HUABIO; 1:1000), anti-p-CHK1 (AF3008, Affinity Biosciences; 1:1000), anti-p-CHK2 (AF3036, Affinity Biosciences; 1:1000), anti-CDK1 (DF6024, Affinity Biosciences; 1:1000), anti-CDC25C (AF6258, Affinity Biosciences; 1:1000), anti-p-P53 (AF3075, Affinity Biosciences; 1:1000), and anti-GAPDH (81640-5-RR, Proteintech; 1:1000). After washing three times with TBST (10 minutes each time), the membrane was incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibody (Proteintech; 1:5000) at room temperature for 1 hour. Finally, the membrane was developed using BeyoECL Plus Western Blotting assay solution (P0018S, Beyotime), and images were acquired using a ChemiDoc MP imaging system (Bio-Rad).

[0061] To elucidate the mechanism by which the small molecule conjugate IX mediates radiosensitization, we assessed the degree of DNA damage using immunofluorescence staining at the γH2AX focal spot. Figure 6 As shown, single-agent treatment with small molecule conjugate IX induced γH2AX focus formation in HepG2 and Huh-7 cells in a dose-dependent manner. For example, in HepG2 cells, the average number of γH2AX focuses per nucleus increased from 2.6 in the control group to 13.4 and 19.4 after treatment with 2.5 μM and 5 μM small molecule conjugate IX, respectively. Notably, pre-incubation of small molecule conjugate IX 24 hours before irradiation synergistically enhanced γH2AX focus formation compared to treatment with small molecule conjugate IX alone or irradiation alone.

[0062] Example 9: This embodiment describes an in vivo tumor suppression experiment on the small molecule conjugate compound IX, which is synergistic with low-dose (2 Gy) radiotherapy.

[0063] To evaluate whether the radiosensitizing effect of the small molecule conjugate compound IX could enhance the in vivo radiotherapy response, we further investigated its antitumor effect in combination with low-dose (2 Gy) radiotherapy. Specifically, in a precise in vivo chemoradiotherapy paradigm, female BALB / c-nude nude mice were subcutaneously inoculated with HepG2 cells (5 × 10⁻⁶) in the right hind limb. 6 (cells). When the tumor reaches 100-200 mm 3 Mice were randomly assigned to a saline control group, a 10 mg / kg compound IX monotherapy group, a single radiotherapy group (2 Gy), and a combination group receiving 10 mg / kg small molecule conjugate IX plus radiotherapy (2 Gy). Mice received intravenous injections of 10 mg / kg small molecule conjugate IX twice weekly for four weeks, followed by radiotherapy the next day, with each injection receiving 2 Gy. During radiotherapy, only the hind limb tumors were exposed, while other parts of the body were shielded with lead. Mouse weight and tumor size were measured every two days. Tumor volume was calculated using the formula: Tumor size = 1 × w 2 / 2( l Major axis; W (Short diameter). Tumor size-time curves and body weight-time curves were plotted using GraphPad Prism 5.0. After treatment, mice were sacrificed, and tumor tissue was harvested for γH2AX immunohistochemical staining. The heart, liver, spleen, lungs, and kidneys of the mice were also subjected to H&E staining.

[0064] To evaluate the in vivo radiosensitization potential of the small molecule conjugate compound IX, we investigated its antitumor efficacy in combination with low-dose irradiation (2 Gy) using the HepG2 xenograft model. Figure 7 As shown, mice were treated twice weekly with the small molecule conjugate compound IX (10 mg / kg) followed by 2 Gy irradiation for 4 weeks. Tumor growth was monitored and treatment efficacy was assessed on day 27. Despite the use of a low dose of the small molecule conjugate compound IX, the combination therapy significantly delayed tumor growth (final volume: 22 mm³), which was superior to the solvent control group (1222 mm³). P < 0.0001), small molecule conjugate IX monotherapy group (526 mm³; P < 0.01) or irradiation alone (257 mm³); P < 0.01), such as Figure 8As shown, the combination therapy effectively induced tumor regression, achieving a tumor growth inhibition (TGI) rate of 98%. Importantly, no significant weight loss was observed during the study, indicating that the treatment was well-tolerated.

[0065] Safety assessments further support the superiority of this combination regimen. Hematoxylin and eosin (H&E) staining of major organs (heart, liver, spleen, lungs, and kidneys) did not show significant treatment-related pathological changes, such as... Figure 9 As shown. Mechanistic studies of tumor tissue obtained on day 27 revealed that the combination therapy group exhibited enhanced DNA damage response, with a significantly higher frequency of γH2AX-positive cells compared to either single-drug therapy group. Figure 10 As shown in the figure, this indicates that the small molecule conjugate IX can serve as a tumor-selective radiosensitizer, synergistically improving tumor control with radiotherapy, making it a promising candidate for precision chemoradiotherapy in HCC. Notably, the combination therapy further enhanced the expression of CALR and HMGB1, exceeding the levels achieved by either treatment alone. This strong induction of the in vivo ICD marker suggests that the small molecule conjugate IX not only induces ICD but also synergistically amplifies this immunogenic response with radiotherapy. This dual activity highlights the potential of the small molecule conjugate IX in both chemoradiotherapy modalities for HCC.

[0066] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A small molecule coupling compound targeting ASGPR, with the following general formula: ； in, X is a small molecule ligand with the ability to target ASGPR; Y is a derivative of cucurbitacin B.

2. The small molecule coupling compound targeting ASGPR according to claim 1, characterized in that, The small molecule ligand with ASGPR targeting capability is a carbohydrate small molecule ligand, and the carbohydrate small molecule in the carbohydrate small molecule ligand is... N - Acetylgalactose, lactose, lactoside N - One of acetylglucosamines.

3. The small molecule coupling compound targeting ASGPR according to claim 2, characterized in that, The carbohydrate ligands in the carbohydrate ligands are trimers.

4. The small molecule coupling compound targeting ASGPR according to any one of claims 1 to 3, characterized in that, The structure of the cucurbitacin B derivative is as follows: 。 5. A method for preparing a small molecule coupling compound targeting ASGPR according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step S1: Prepare cucurbitacin B derivatives and small molecule ligands with ASGPR targeting capabilities; Step S2: The prepared cucurbitacin B derivative and the prepared small molecule ligand with ASGPR targeting ability are subjected to a copper-catalyzed click chemical cyclization reaction under CuSO4·5H2O / sodium vitamin C catalysis to obtain the target compound.

6. The method for preparing a small molecule coupling compound targeting ASGPR according to claim 5, characterized in that, In step S1, the specific process for preparing the cucurbitacin B derivative is as follows: using cucurbitacin B as the starting material, it is reacted with 6-azidohexanoic acid under EDCI and DMAP catalytic conditions, and after esterification, compound I is obtained.

7. The method for preparing a small molecule coupling compound targeting ASGPR according to claim 6, characterized in that, In step S1, the small molecule ligand with ASGPR targeting ability prepared is a trimer. N The preparation process of the acetylgalactose small molecule ligand is as follows: using tromethamine as the initial raw material, it undergoes a Mac addition reaction with butyl acrylate under the action of NaOH to obtain compound II; compound II reacts with benzyl chloroformate under the action of Na2CO3 to obtain amino-protected compound III; compound III is detert-butylated under the catalysis of trifluoroacetic acid to obtain compound IV. Compound IV reacted with propynyl tetraglycolic acid under HBTU and HOBT catalysis to give compound V; compound V underwent deCbz protecting group removal under 10% Pd / C and H2 catalysis to give compound VI; Compound VI was reacted with propynyl tetraglycolic acid under HBTU and HOBT catalysis to give compound VII by amidation. Compound VII was deacetylated under NaOMe / MeOH conditions to yield a trimer. N - Acetylgalactose small molecule ligand compound VIII.

8. The method for preparing a small molecule coupling compound targeting ASGPR according to claim 7, characterized in that, In step S2, compound I and compound VIII undergo a copper-catalyzed click cyclization reaction under CuSO4·5H2O / sodium vitamin C catalysis, followed by purification by preparative HPLC to obtain purified small molecule coupling compound IX.

9. The use of a drug that targets ASGPR and has precise chemotherapy and radiosensitizing functions, prepared with the small molecule conjugate of any one of claims 1 to 4 or the small molecule conjugate of any one of claims 5 to 8 and its salts and hydrates as the main components, and the remainder being pharmaceutically acceptable, non-toxic, and non-inert pharmaceutical carriers and / or excipients for humans and animals, characterized in that... The drug, which has the functions of precision chemotherapy and radiosensitizing therapy, can be used to prevent, diagnose and / or treat diseases related to ASGPR expression.

10. The use of the drug with precise chemotherapy and radiosensitizing functions according to claim 9, characterized in that, The prevention, diagnosis, and / or treatment of diseases associated with ASGPR expression include liver cancer and tumors associated with ASGPR expression.

Images