A liver-targeting glycoligand molecule modified with dual antennae GalNAc and its drug delivery system

Abstract

This invention provides a liver-targeting glycoligand molecule modified with dual-antenna GalNAc and its drug delivery system. This liver-targeting glycoligand molecule and its drug delivery system, through ASGPR recognition, can maximize the concentration of therapeutic drugs in liver tumor parenchymal cells, thereby improving the targeting of drug distribution, increasing the therapeutic index, reducing systemic toxicity, and improving patient survival time and quality of life. The liver-targeting glycoligand molecule of this invention is synthesized using an enzymatic synthesis method, which involves fewer synthesis steps, mild enzymatic reaction conditions, high regioselectivity, high reaction efficiency, is environmentally friendly, and has low production costs, making it highly promising for industrialization.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulations, specifically to a liver-targeting glycoligand molecule, particularly a liver-targeting glycoligand molecule targeting the liver ASGPR receptor and its drug delivery system. Background Technology

[0002] Primary liver cancer (PLC) is one of the most common malignant tumors of the digestive tract in clinical practice, with 90% being hepatocellular carcinoma (HCC). Since liver cancer and other liver diseases, such as fibrosis and cirrhosis, primarily affect hepatic parenchymal cells, targeted delivery of therapeutic drugs to these cells is a promising treatment approach.

[0003] However, chemotherapy drugs for treating liver cancer generally suffer from numerous drawbacks, including low efficacy and numerous side effects. Besides the fact that the pharmacological effects of the chemotherapy drugs themselves are not yet ideal, the main reason is that they cannot be accurately and effectively transported to the liver lesions, especially the liver parenchymal cells, resulting in low drug bioavailability and significantly reducing their effectiveness. Therefore, increasing drug concentration at the cancerous site and prolonging the duration of drug action at the cancerous site have gradually become a hot topic in liver cancer treatment research.

[0004] To design a hepatocyte-targeted drug delivery system, we need to find a target receptor that is highly expressed on the surface of hepatocytes but minimally expressed in other cells. The asialoglycoprotein receptor (ASGPR) is a calcium-dependent C-type lectin specifically and abundantly expressed on hepatocytes of all mammals. Studies have shown that glycoproteins or glycoligands ending in Gal or GalNAc residues can be recognized by ASGPR, with GalNAc exhibiting a binding affinity to ASGPR that is tens of times (approximately 50 times) higher than that of Gal [D'Souza, AA, & Devarajan, PV, 2015. Asialoglycoprotein receptor mediated hepatocyte targeting—Strategies and applications. J. Control. Release, 203, 126-139.]. Therefore, ASGPR can serve as a target for liver targeting; however, finding suitable ligand molecules capable of entering hepatocytes remains a significant challenge. Summary of the Invention

[0005] The main objective of this invention is to provide a liver-targeting glycoligand molecule modified with dual-antenna GalNAc and its drug delivery system. This liver-targeting glycoligand molecule and its drug delivery system, through ASGPR recognition, can maximize the concentration of therapeutic drugs in liver tumor parenchymal cells, thereby improving the targeting of drug distribution, increasing the therapeutic index, reducing systemic toxicity, and improving patient survival time and quality of life. Furthermore, the liver-targeting drug delivery system can protect the loaded drug from enzymatic degradation in the body, improving stability, while also increasing drug solubility in water and extending the drug's half-life. This liver-targeting drug delivery system can not only load chemotherapy drugs for treating liver cancer, but also other drugs for treating fatty liver, cirrhosis, viral hepatitis, and DNA-related liver diseases, greatly enhancing the therapeutic effect of existing drugs and thus improving patient survival time and quality of life.

[0006] Specifically, one objective of this invention is to provide a liver-targeting glycoligand molecule modified with dual-antennae GalNAc, the general structural formula of which is as follows:

[0007]

[0008] Wherein, R1 = CH3CONH- or -OH, R2 = CH2 or CH2OCH2, and R3 = CH2. n = 1-9. Preferably, n = 1-6. Preferably, R1 = CH3CONH-, and R2 or R3 = CH2.

[0009] In one specific embodiment of the present invention, the molecular structure of the liver-targeting glycoligand is as follows:

[0010]

[0011] One of the objectives of this invention is to provide a drug delivery system containing the aforementioned dual-antennae GalNAc-modified liver-targeting glycoligand molecule, the drug delivery system comprising:

[0012]

[0013] The phospholipids and their structural analogues include, but are not limited to, lecithin, soybean phospholipids, etc.

[0014] Furthermore, the drug delivery system includes:

[0015]

[0016] Preferably, the drug delivery system includes:

[0017]

[0018] Preferably, the drug delivery system further includes other pharmaceutically acceptable excipients.

[0019] In the preferred embodiment of the present invention, the drugs include, but are not limited to, doxorubicin, epirubicin, paclitaxel, docetaxel, cisplatin, vincristine, lenvatinib, siRNA, and other drugs for treating liver diseases.

[0020] One of the objectives of this invention is to provide the use of liver-targeting glycoligand molecules containing the above-mentioned dual-antennae GalNAc modification and their drug delivery systems in the preparation of drugs for treating liver diseases.

[0021] One objective of this invention is to provide a method for treating liver diseases using a liver-targeting glycoligand molecule containing the aforementioned dual-antenna GalNAc-modified drug and its drug delivery system. The method includes administering a subject a therapeutically effective amount of the drug as described in this invention, delivered into the subject's body via the dual-antenna GalNAc-modified liver-targeting glycoligand molecule and its drug delivery system.

[0022] The liver diseases mentioned include, but are not limited to, liver cancer, cirrhosis, viral hepatitis, and liver fibrosis.

[0023] One of the objectives of this invention is to provide a method for preparing the above-mentioned dual-antennae GalNAc-modified liver-targeting glycoligand molecule, wherein the preparation method is carried out through bio-enzymatic synthesis, and the reaction route is as follows:

[0024]

[0025] The bioenzyme is selected from one or more of Novozym 435, PSIM, CRL, and TL IM; the reaction temperature is 10-70℃, preferably 30-60℃; the reaction solvent is an organic solvent, including but not limited to one or more of isooctane, tetrahydrofuran, n-hexane, acetone, tert-butanol, benzene, toluene, and pyridine, and combinations thereof.

[0026] Specifically, the reaction route is as follows:

[0027]

[0028] In the preferred embodiment of the present invention, the reaction solvent in step (1) is selected from one or more of isooctane, tetrahydrofuran, n-hexane, acetone, tert-butanol, benzene, toluene, and pyridine, or a combination thereof; the substrate molar ratio is 1:1-1:10, preferably 1:2-1:6.

[0029] In the preferred embodiment of the present invention, the reaction solvent in step (2) is selected from one or more of tetrahydrofuran, tert-butanol, acetonitrile, benzene, toluene, and pyridine, or a combination thereof; the substrate molar ratio is 1:1-1:10, preferably 1:1-1:4.

[0030] In the preferred embodiment of the present invention, the reaction solvent in step (3) is selected from one or more of tetrahydrofuran, acetone, tert-butanol, acetonitrile, benzene, toluene, and pyridine, or a combination thereof; the substrate molar ratio is 1:1-1:15, preferably 1:2-1:8.

[0031] In the preferred embodiment of the present invention, the reaction solvent in step (4) is selected from one or more of acetone, tert-butanol, tetrahydrofuran, acetonitrile, benzene, toluene, and pyridine, or a combination thereof; the substrate molar ratio is 1:1-1:30, preferably 1:4-1:16.

[0032] The enzymatic synthesis method for ligand molecules described in this invention has significant advantages: fewer synthesis steps, inexpensive and readily available raw materials, mild enzymatic reaction conditions, high regioselectivity, high reaction efficiency, green and environmentally friendly, low production cost, and great industrialization potential.

[0033] This invention includes a bilingual (English and Chinese) explanation and abbreviations.

[0034] Attached Figure Description

[0035] Figure 1 Example 10: Results of HepG2 cell uptake experiment;

[0036] Figure 2 Example 10: Results of HepG2 cell uptake inhibition assay, specifically the uptake of DiR-Unmod-LP, DiR-LP-6-GalNAc, DiR-LP-PEG2-6-GalNAc, and DiR-LP-6-(GalNAc)2 by HepG2 cells. **: P < 0.01, *: P < 0.5; ##: P < 0.01, DiR-LP-6-(GalNAc)2 compared with other groups; $$: P < 0.01, $: P < 0.5, DiR-LP-PEG2-6-GalNAc and DiR-LP-6-GalNAc groups compared with DiR-Unmod-LP. (n = 3).

[0037] The present invention will be further described below through specific embodiments. Detailed Implementation

[0038] The following specific embodiments illustrate the present invention. It should be noted that these specific embodiments are only for further explanation and do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the scope of protection of the present invention.

[0039] Instruments: AB 4000QTRAP LC-MS / MS triple quadrupole liquid chromatography-mass spectrometry system (SCIEX, USA); Bruker 500M nuclear magnetic resonance spectrometer (Bruker, Switzerland); N-1100D-WB rotary evaporator (Tokyo RIKEN, Ltd.); Sartorius 125D electronic balance (Sartorius, Germany); Nexera XR high performance liquid chromatograph (Shimadzu Enterprise Management (China) Co., Ltd.); constant temperature shaker (Taicang Experimental Equipment Factory); MS7-H550-Pro magnetic stirrer (Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd.); SX2-4-10N box muffle furnace (Shanghai Qixin Scientific Instruments Co., Ltd.).

[0040] Reagents: Novozym 435 (derived from Candida antarctica B, immobilized on macroporous anion exchange resin), TL IM (derived from Thermomyces lanuginosus, immobilized on macroporous anion exchange resin), and RM IM (derived from Rhizomucor miehei, immobilized on macroporous anion exchange resin) were all purchased from Novozymes; PSIM (derived from Burkholderia cepacia, immobilized on diatomaceous earth) and lipase AYS (derived from Candida rugosa) were purchased from Amano; molecular sieves (Aladdin Biochemical Technology Co., Ltd.)

[0041] The synthetic route for the glycoligand molecule of this invention is as follows:

[0042]

[0043] Example 1: Synthesis of (5-cholesten-yl)vinyl adipic acid

[0044] Take a stoppered conical flask, weigh adipic acid divinyl ester and cholesterol, and add an appropriate amount of dehydrating solvent (to... Molecular sieves were activated at 350℃ for 4 hours, cooled in a desiccator, added to an organic solvent, and shaken at room temperature for 48 hours. The molecular sieves were then filtered off, yielding a dehydrated solvent. This solvent was then shaken in a constant-temperature shaker for 30 minutes, and enzyme was added to initiate the reaction. After the reaction was complete, the enzyme was removed by filtration, and the filtrate was vacuum-dried to obtain a viscous liquid. This liquid was then dissolved in an appropriate amount of methanol by sonication, allowed to stand at 0℃ for 24 hours to crystallize, and then vacuum-filtered at low temperature to obtain a white powder. The synthetic route is shown in the figure below.

[0045]

[0046] Identification of Synthetic Products

[0047] MS conditions: The product was dissolved in an appropriate amount of methanol and analyzed by mass spectrometry (MS). Mass spectrometry parameters: triple quadrupole LC-MS / MS: electrospray ionization (ESI+) detection, scan range m / z 150-m / z 1000. (The same applies below)

[0048] The product was identified by MS and NMR, and the results are as follows: MS: [M+H] + =541.8. 1 H NMR(500MHz,Pyr)δ7.49(H-34,dd,J=14.0,6.3Hz,1H),5.43–5.40(H-9,m,1 H),4.95(H-35a,dd,J=14.0,1.5Hz,1H),4.85(H-35b,m,1H),4.58(H-2,dd, J=6.3,1.5Hz,1H),2.47(m,6H),1.58(m,30H),1.03(H-19,s,3H),1.00(H-2 7,d,J=6.6Hz,3H),0.93(H-24,25,dd,J=6.6,1.2Hz,6H),0.70(H-26,s,3H). 13 C NMR(126MHz,Pyr)δ172.98(C-28),170.92(C-33),142.16(C-34),140.46(C-6),123.27(C-9),97.93(C-35),74.41(C-2),57.25(C -14),56.84(C-15),50.71(C-7),42.95(C-21),40.40(C-12),40.20(C-22),38.99(C-4),37.68(C-3),37.27(C-5),36.96(C-18),

[0049] 36.50(C-20),34.72(C-29),33.97(C-32),32.60(C-10),32.49(C-8),28.95(C-16),28.70(C-23),28.59(C-1),25.10(C-31),2 4.95(C-30),24.71(C-17),24.63(C-21),23.39(C-24),23.15(C-25),21.73(C-11),19.82(C-27),19.41(C-19),12.45(C-26).

[0050] Depend on 13According to the C NMR spectrum, δ172.98 and 170.92 are characteristic peaks of carbonyl carbon, and the shift values ​​are different, indicating that the two carbonyl carbon atoms are in different chemical environments. At the same time, characteristic peaks of the carbon of the ethylene ester double bond were also observed: δ142.16 and 97.93

[23] . We speculate that only one side of the ethylene ester group on both sides of adipic acid divinyl ester is substituted; δ140.46 and 123.27 are the C-6 and C-9 signal peaks in the carbon-carbon double bond in the upper ring of cholesterol, respectively

[24] ; the C-2 shift value of cholesterol is δ71.81 (SDBSWeb:

[0051] (https: / / sdbs.db.aist.go.jp), after the OH group attached to it is esterified, the signal peak of C-2 shifts to the lower field by about 3 ppm, which is basically consistent with the actual observed value of 74.41.

[0052] Example 2: Optimization of the synthesis process of (5-cholesten-yl)vinyl adipic acid

[0053] Using the yield of (5-cholesten-yl)vinyl adipic acid as an indicator, the effects of enzyme type, substrate molar ratio, solvent type and reaction temperature on the synthesis yield were investigated sequentially using a single-factor rotation method.

[0054] Chromatographic conditions: Column: Hypersil ODS-2 column (250 mm × 4.6 mm, 5 μm, Thermo Scientific); Wavelength: 195 nm; Mobile phase: pure methanol, isocratic elution; Injection volume: 10 μL; Column temperature: 35 ℃; Flow rate: 1.0 mL / min.

[0055] (1) Types of enzymes

[0056] Reaction conditions: 20mg cholesterol, 40mg adipic acid divinyl ester, 50mg Molecular sieves, 5 mL of dehydrated isooctane, and 10 U of different enzymes (Novozym 435, PSIM, RM IM, TLIM, AYS) were used in a constant temperature shaker at 45℃ and 200 r / min. The yields at 0, 1, 2, 3, 4, 5, 6, 7, 16, and 24 h were determined by HPLC. The results are shown in the table below.

[0057]

[0058] (2) Substrate molar ratio

[0059] Reaction conditions: 20mg cholesterol, wherein cholesterol: adipic acid divinyl ester = (1:2, 1:3, 1:4, 1:5, 1:6), 50mg Molecular sieve, 5 mL of dehydrated isooctane, 10 U of PSIM, and reaction were carried out in a constant temperature shaker at 45 °C and 200 r / min. The yields at 0, 1, 2, 3, 4, 5, 12, and 24 h were determined by HPLC. The results are shown in the table below.

[0060]

[0061] (3) Types of solvents

[0062] Reaction conditions: 20mg cholesterol, 100mg adipic acid divinyl ester, 50mg Molecular sieves were reacted in a constant temperature shaker at 45℃ and 200 r / min with 5 mL of different dehydrated solvents (isooctane, tetrahydrofuran, n-hexane, acetone, and tert-butanol) and 10 U of PSIM. The yields at 0, 1, 2, 3, 4, 5, 12, and 24 h were determined by HPLC. The results are shown in the table below.

[0063]

[0064] (4) Investigation of enzyme dosage

[0065] Reaction conditions: 20mg cholesterol, 100mg adipic acid divinyl ester, 50mg Molecular sieves were added to 5 mL of dehydrated n-hexane and different masses of PSIM. The reaction was carried out in a constant temperature shaker at 45℃ and 200 r / min. The yields at 0, 1, 2, 3, 4, 5, 12, and 24 h were determined by HPLC. The results are shown in the table below.

[0066]

[0067] (5) Reaction temperature

[0068] Reaction conditions: 20mg cholesterol, 100mg adipic acid divinyl ester, 50mg Molecular sieves were added to 5 mL of dehydrated n-hexane and 5 U PSIM, and the mixtures were reacted in constant temperature shakers (200 r / min) at 35 °C, 45 °C, and 55 °C, respectively. The yields at 0, 1, 2, 3, 4, 5, 12, and 24 h were measured by HPLC, and the results are shown in the table below.

[0069]

[0070] Example 3 Synthesis of Chol-(OH)2

[0071] Accurately weigh 1.62 g of N-(3-aminopropyl)diethanolamine and 5.42 g of (5-cholesten-yl)vinyladipic acid, and dissolve them in 20 mL and 5 mL of THF solvent, respectively, by stirring thoroughly. Place the N-(3-aminopropyl)diethanolamine solution into a constant-pressure dropping funnel, connect it to a three-necked flask filled with (5-cholesten-yl)vinyladipic acid solution, and purge the entire reaction apparatus with nitrogen. Simultaneously, open the valve of the constant-pressure dropping funnel while starting the magnetic stirrer (600 rpm), and add the N-(3-aminopropyl)diethanolamine solution dropwise at a rate of 2 drops / s. After the addition is complete, remove the constant pressure dropping funnel, seal the flask and place it in a constant temperature shaker (45℃, 200r / min) for reaction. After the reaction is complete, remove the solvent by vacuum rotary evaporation of the reaction solution. Dissolve the residue in a small amount of methanol, use ammonia-methanol (0.7:99.3) as the eluent, and elute with C18 as the packing material to obtain the product.

[0072]

[0073] Identification of Synthetic Products

[0074] The product was identified by MS and NMR, and the results are as follows: MS: [M+H] + =659.7, [M+Na] + =682.4, [2M+H] + =1318.5,[2M+Na] + =1341.0. 1H NMR(500MHz,Pyr)δ8.41(NH,t,J=5.3Hz,1H),5.40–5.36(H-9,m,1H),4.86–4.75(H-2,m,1H),3.88(H-39,H-40, t,J=5.7Hz,4H),3.59(H-34,dd,J=12.3,6.3Hz,2H),2.79(H-37,H-38,t,J=5.7Hz,4H),2.71(H-36,t,J=6.6Hz, 2H),2.51–2.36(H-29,H-32,H-4,m,6H),2.13(s,1H),2.07–1.72(m,11H),1.72–1.35(m,10H),1.32–1.02(m,10 H), 1.00 (H-27, s, 3H), 0.98 (H-19, d, J = 6.5 Hz, 3H), 0.90 (H-24, H-25, dd, J = 6.6, 1.2 Hz, 6H), 0.68 (H-26, s, 3H). 13 C NMR(126MHz,Pyr)δ173.20(C-28),173.03(C-33),140.43(C-6),123.17(C-9),74.26(C-2),60.74(C-39,C-40),57.68(C-37,C-38),57.18(C- 14),56.77(C-15),54.08(C-36),50.64(C-7),42.89(C-21),40.34(C- 12),40.14(C-22),38.93(C-4),38.58(C-34),37.62(C-3),37.20(C-5) ,36.89(C-18),36.62(C-32),36.43(C-20),34.95(C-29),32.53(C-10 ),32.43(C-8),28.89(C-16),28.63(C-23),28.54(C-1),28.07(C-35), 26.13(C-31),25.56(C-30),24.89(C-17),24.56(C-21),23.34(C-24), 23.09(C-25),21.66(C-11),19.77(C-27),19.35(C-19),12.39(C-26).

[0075] Example 4: Optimization of the synthesis process of Chol-(OH)2

[0076] Using the synthesis yield of Chol-(OH)2 as an indicator, the effects of three factors on the synthesis yield were investigated sequentially using a single-factor rotation method: solvent type (tetrahydrofuran, tert-butanol), substrate molar ratio (1:1, 1:1.5, 1:2, 1:3, 1:4), and reaction temperature (35℃, 45℃, 55℃).

[0077] Chromatographic conditions: Column: Hypersil ODS-2 column (250 mm × 4.6 mm, 5 μm, Thermo Scientific); Mobile phase: methanol: 1% ammonia = 95:5, isocratic elution; Injection volume: 10 μL; Column temperature: 40℃; Flow rate: 1.0 mL / min; UNIEX-7700 evaporative light detector; Drift tube temperature: 50℃; Carrier gas flow rate: 20 psi.

[0078] (1) Solvent types

[0079] Reaction conditions: 270.21 mg of (5-cholesten-yl)vinyl adipic acid and 81.11 mg of N-(3-aminopropyl)diethanolamine were added to 20 mL and 5 mL of different solvents (tetrahydrofuran and tert-butanol), respectively, and stirred thoroughly to dissolve. The reaction was started under the conditions described in Example 3. Reaction solutions were collected at 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours and analyzed by HPLC. The results are shown in the table below.

[0080]

[0081] (2) Substrate molar ratio

[0082] Reaction conditions: 270.21 mg (0.5 mmol) of (5-cholesten-yl)vinyl adipic acid and N-(3-aminopropyl)diethanolamine were weighed at molar ratios of 1:1, 1:1.5, 1:2, 1:3, and 1:4, respectively, and 5 mL and 20 mL of tert-butanol were added to each mixture and stirred thoroughly to dissolve. The reaction was started under the conditions described in Example 3. Reaction solutions were collected at 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours and analyzed by HPLC. The results are shown in the table below.

[0083]

[0084] (3) Reaction temperature

[0085] Reaction conditions: 270.21 mg of (5-cholesten-yl)vinyl adipic acid and 324.44 mg of N-(3-aminopropyl)diethanolamine were dissolved in 5 mL of tert-butanol and 20 mL of tert-butanol, respectively, by thorough stirring. The reaction was started under the conditions described in Example 3, with the temperature of the isothermal shaker set to 35°C, 45°C, and 55°C. Reaction solutions were collected at 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours, respectively, and analyzed by HPLC. The results are shown in the table below.

[0086]

[0087] Example 5: Synthesis of GalNAc-6-adipic acid divinyl ester

[0088] Accurately weigh 0.1 mmol of acetylgalactosamine (GalNAc) and 0.2 mmol of adipic acid divinylester. 1 g of molecular sieve and 10 mL of THF were added to a stoppered conical flask and placed in an air bath shaker (45℃, 200 r / min) for 30 min. Then, Novozym 435 enzyme was added to initiate the reaction. After the reaction was complete, the reaction solution was filtered to remove the enzyme. The filtrate was dried under vacuum to obtain a white solid, which was then dissolved in an appropriate amount of methanol upon heating. The solid was recrystallized at -20℃ for 24 h, filtered again, and the residue was dried under vacuum to obtain the product.

[0089]

[0090] Identification of Synthetic Products

[0091] The product was identified by MS and NMR, and the results are as follows: MS: [M+H] + =376.2, [M+Na] + =397.9; 1H NMR(500MHz,Pyr)δ8.68(NH,d,J=8.6Hz,1H),7.44(H7,ddd,J=14.0,6.3,1.8Hz,1H),5.94(H1',s,1H), 5.33–5.23(H2',m,1H),4.92(H8'a,dd,J=14.0,1.5Hz,1H),4.88–4.81(H6'a,H5',m,2H),4.79(H6'b,dd ,J=8.7,3.2Hz,1H),4.60(H3',dd,J=11.0,3.3Hz,1H),4.54(H8'b,dd,J=6.3,1.5Hz,1H),4.44(H4',d, J=2.9Hz,1H),2.36–2.24(H2,H5,m,4H),2.10(H8',d,J=7.7Hz,3H),1.60(H3,H4,td,J=7.4,3.9Hz,4H); 13 C NMR(126MHz,Pyr)δ173.46(C-1),171.41(C-7'),170.83(C-6),142.05(C-7),97.91(C-8),93.23(C-1'),70.74(C-4'),69 .85(C-3'),69.45(C-5'),65.65(C-6'),52.53(C-2'),34.19(C-2),33.80(C-5),24.80(C-3),24.55(C-4),23.60(C-8').

[0092] GalNAc-6-adipic acid divinyl ester, generated by transesterification of adipic acid divinyl ester with a hydroxyl donor on GalNAc, is produced by... 13 CNMR analysis was used to determine the esterification site of the sugar. The carbon NMR spectrum of GalNAc-6-adipic acid divinyl ester showed a similarity to that of GalNAc standard. 13 Compared with CNMR data, the chemical shift of C-6 in GalNAc in the product shifted from δ63.26 to δ65.65 at a lower field, while the adjacent C-5 chemical shift shifted from δ72.48 (unsubstituted) to δ69.45 at a higher field; the chemical shifts of carbons at other positions did not change significantly. 13 CNMR analysis showed that the transesterification reaction between adipic acid divinyl ester and GalNAc occurred at the C-6 hydroxyl group.

[0093] Example 6: Optimization of the synthetic process of GalNAc-6-adipic acid divinyl ester

[0094] Using the yield of GalNAc-6-adipic acid divinyl ester as an indicator, the effects of enzyme type (Novozym 435, PSIM, CRL, TL IM), substrate molar ratio (1:2, 1:4, 1:6, 1:8), solvent type (tetrahydrofuran, acetone, tert-butanol), and reaction temperature (35℃, 45℃, 55℃) on the synthesis yield were investigated using single-factor methods.

[0095] Chromatographic conditions: Column: Hypersil ODS-2 column (250 mm × 4.6 mm, 5 μm, Thermo Scientific); wavelength: 210 nm; mobile phase: methanol (A)-water (B); gradient elution (0–3 min 10% A; 3–6 min 10% → 30% A; 6–16 min 30% → 40% A; 16–19 min 40% → 100% A; 19–24 min 100% A; 24–27 min 100% → 10% A; 27–35 min 10% A); injection volume: 10 μL; column temperature: 40 °C; flow rate: 1.0 mL / min

[0096] (1) Types of enzymes

[0097] Reaction conditions: GalNAc 22.1 mg, adipic acid divinyl ester 39.6 mg, 1 g of molecular sieve and 10 mL of tetrahydrofuran were added to a stoppered conical flask and placed in an air bath shaker (45℃, 200 rpm) for 30 min. Then, 160 U of different enzymes (TL IM, PSIM, CRL, Novozym 435) were added to initiate the reaction. Samples of 200 μL were taken at 0, 1, 2, 4, 6, 8, 12, and 24 h, and centrifuged at 10000 rpm for 10 min in a refrigerated centrifuge. 100 μL of the supernatant was diluted with 900 μL of methanol and analyzed by HPLC. The results are shown in the table below.

[0098]

[0099] (2) Types of solvents

[0100] Reaction conditions: GalNAc 22.1 mg, adipic acid divinyl ester 39.6 mg, 1 g of molecular sieve was added in fractions of 10 mL each of different solvents (tetrahydrofuran, acetone, and tert-butanol) to a stoppered conical flask. The flask was then placed in an air bath with a shaker (45℃, 200 rpm) and shaken for 30 min. Novozym 435 80 U was then added to initiate the reaction. Samples of 200 μL were taken at 1, 2, 4, 6, 8, 12, and 24 h, and centrifuged at 10000 rpm for 10 min. 100 μL of the supernatant was diluted with 900 μL of methanol and analyzed by HPLC. The results are shown in the table below.

[0101]

[0102]

[0103] (3) Enzyme dosage

[0104] Reaction conditions: GalNAc 22.1 mg, adipic acid divinyl ester 39.6 mg, 1 g of molecular sieve and 10 mL of tetrahydrofuran were added to a stoppered conical flask and placed in an air bath shaker (45℃, 200 rpm) for 30 min. Different masses of Novozym 435 (40, 80, 120, and 160 U) were then added to initiate the reaction. 200 μL samples were taken at 1, 2, 4, 6, 8, 12, and 24 h, centrifuged at 10000 rpm for 10 min in a refrigerated centrifuge, and 100 μL of the supernatant was diluted with 900 μL of methanol and analyzed by HPLC. The results are shown in the table below.

[0105]

[0106] (4) Substrate molar ratio

[0107] Reaction conditions: Weigh 22.1 mg GalNAc, adipic acid divinyl ester: GalNAc in ratios of 2:1, 4:1, 6:1, or 8:1 (molar ratio). 1 g of molecular sieve and 10 mL of tetrahydrofuran were added to a stoppered conical flask and placed in an air bath shaker (45℃, 200 rpm) for 30 min. Then, 160 U of Novozym 435 was added to start the reaction. Samples of 200 μL were taken at 0, 1, 2, 4, 6, 8, 12, and 24 h, and centrifuged at 10000 rpm for 10 min in a refrigerated centrifuge. 100 μL of the supernatant was diluted with 900 μL of methanol and analyzed by HPLC. The results are shown in the table below.

[0108]

[0109] (5) Reaction temperature

[0110] Reaction conditions: GalNAc 22.1 mg, adipic acid divinyl ester 158.5 mg, 1 g of molecular sieve and 10 mL of tetrahydrofuran were added to a stoppered conical flask and placed in an air bath constant temperature shaker (200 r / min) at different temperatures (35℃, 45℃, 55℃) for 30 min. Then, 160 U of Novozym 435 was added to start the reaction. 200 μL samples were taken at 0, 1, 2, 4, 6, 8, 12, and 24 h, and centrifuged at 10000 r / min for 10 min in a refrigerated centrifuge. 100 μL of the supernatant was diluted with 900 μL of methanol and analyzed by HPLC. The results are shown in the table below.

[0111]

[0112] Example 7: Synthesis of Chol-6-(GalNAc)2

[0113] Weigh 2.66 g Chol-(OH)2, 1.50 g GalNAc-6-adipic acid divinyl ester, 1.00 g molecular sieve, and 50 mL THF. Add them to a stoppered conical flask and place it in a constant temperature air bath shaker at 200 r / min. Shake at 45 °C for 30 min, then add 0.4 g Novozyme 435. Seal the reaction for 24 h. Filter the reaction solution to remove the enzyme, and evaporate the filtrate under vacuum. Dissolve the residue in ether, let it stand at 0 °C for 5 h, and recrystallize. Filter to obtain a white solid, which is the product.

[0114]

[0115] Identification of Synthetic Products

[0116] The product was identified by MS and NMR, and the results are as follows: MS-ESI: [M+H] + =1323.1,[M+Na] + =1345.2; 1H NMR(500MHz,Pyr)δ8.67(N’-H,d,J=8.5Hz,2H),8.30(N-H,t,J=5.5Hz,1H),5.95(H1’,H1”,d,J=3.1Hz,2H),5.41(H-9,d,J=5.0Hz,1H),5.30–5.23(H2’,H2”,m,2H),4.86(H6’a,H5’,H6”a,H5”,H-2,ddd,J=9.9,7.8,6.3Hz,5H),4.80(H6’b,H6”bdd,J=8.7,3.2Hz,2H),4.64–4.57(H3’,H3”,m,1H),4.46(H4’,H4”,d,J=2.6Hz,1H),4.26(H-39,H-40,t,J=6.0Hz,4H),3.54(H-34,dd,J=12.7,6.6Hz,2H),2.78(H-37,H-38,t,J=6.0Hz,4H),2.65(H-36,t,J=6.9Hz,2H),2.45(H-29,H-32,H-4,t,J=7.3Hz,6H),2.39–2.32(H45,H42,H48,H51,m,8H),2.13(H8’,H8”,d,J=6.2Hz,3H),2.04–1.04(m,53H),1.02(H-27,s,3H),1.00(H-19,d,J=6.5Hz,3H),0.92(H-24,H-25,dd,J=6.6,1.2Hz,6H),0.69(H-26,s,3H); 13C NMR(126MHz,Pyr)δ173.56(C-41,C-46,C-47,C-52),173.22(C-28),173.07(C-33),171.41(C-7',C- 7"),140.42(C-6),123.17(C-9),93.22(C-1',C-1"),74.27(C-2),70.73(C-4',C-4"),69.85(C-3', C-3”),69.45(C-5’,C-5”),65.63(C-6’,C-6”),63.05(C-39,C-40),57.16(C-14),56.74(C-15),53. 54(C-37,C-38),53.27(C-36),52.52(C-2',C-2”),50.62(C-7),42.87(C-21),40.31(C-12),40.11( C-22),38.92(C-4),38.21(C-34),37.60(C-3),37.19(C-5),36.86(C-5),36.65(C-32),36.40(C-20 ),34.93(C-29),34.31(C-42,45,48,51),32.52(C-10),32.41(C-8),28.87(C-16),28.61(C-23),28 .53(C-1,35),26.11(C-31),25.54(C-30),25.02(C-43,44,49,50),24.86(C-17),24.52(C-21),23. 61(C-8',8”),23.31(C-24),23.06(C-25),21.65(C-11),19.75(C-27),19.33(C-19),12.37(C-26).

[0117] Example 8: Optimization of the synthesis process of Chol-6-(GalNAc)2

[0118] Using the synthesis yield of Chol-6-(GalNAc)2 as an indicator and Novozym 435 as the synthase, the effects of substrate molar ratio (1:4, 1:8, 1:16), solvent type (tetrahydrofuran, acetone, tert-butanol), and reaction temperature (35℃, 45℃, 55℃) on the synthesis yield were investigated using single-factor methods.

[0119] Chromatographic column: XBridge-C18 column (4.6 mm × 150 mm, 3.5 μm); detector: UV detector; wavelength: 210 nm; mobile phase: methanol (containing 0.02% saturated ammonia): water = 95:5, isocratic elution; column temperature: 40 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL; elution time: 15 min.

[0120] (1) Substrate molar ratio

[0121] Reaction conditions: 16.5 mg of Chol-(OH)₂, GalNAc-6-adipic acid divinyl ester:Chol-(OH)₂ = 1:4, 1:8, and 1:16 (molar ratio), 1.00 g of molecular sieve, and 10 mL of acetone were added to a stoppered conical flask and placed in a constant temperature air bath shaker at 200 rpm. After shaking at 45 °C for 30 min, Novozyme 435 80 U was added to start the reaction. 200 μL of the reaction solution was taken at 1, 2, 4, 6, 8, 12, and 24 h and analyzed by HPLC. The results are shown in the table below.

[0122]

[0123] (2) Enzyme dosage

[0124] Reaction conditions: 16.5 mg of Chol-(OH)₂, 37.5 mg of GalNAc-6-adipic acid divinyl ester, 1.00 g of molecular sieve, and 10 mL of acetone were added to a stoppered conical flask and placed in a constant temperature air bath shaker at 200 rpm. After shaking at 45 °C for 30 min, Novozyme 435 (40, 80, 120, and 160 U) was added to initiate the reaction. 200 μL of the reaction solution was taken at 1, 2, 4, 6, 8, 12, and 24 h and analyzed by HPLC. The results are shown in the table below.

[0125]

[0126] (3) Reaction temperature

[0127] Reaction conditions: 16.5 mg of Chol-(OH)₂, 37.5 mg of GalNAc-6-adipic acid divinyl ester, 1.00 g of molecular sieve, and 10 mL of acetone were added to a stoppered conical flask and placed in a constant-temperature air bath shaker at 200 rpm. The mixture was shaken for 30 min at reaction temperatures of 35℃, 45℃, and 55℃, respectively, and then Novozyme 435 160 U was added to initiate the reaction. 200 μL of the reaction solution was collected at 1, 2, 4, 6, 8, 12, and 24 h and analyzed by HPLC. The results are shown in the table below.

[0128]

[0129] Example 9: Preparation of Liposome Drug Delivery System

[0130] DiR-labeled liposomes were prepared using a thin-film dispersion method. The amount of DiR added was 1% (molar ratio) of the total lipids. Each lipid component (see table below) and DiR were dissolved in chloroform and placed in a rotary evaporator at 55°C to evaporate the chloroform until a lipid film was formed on the inner wall of the flask. The mixture was then vacuum dried for 1 hour, followed by the addition of 10 mL of distilled water and incubation at 55°C for 1 hour. The mixture was then extruded 10 times each through 0.2, 0.1, and 0.05 μm Track-Etched Polycarbonate Membranes in a constant temperature water bath at 65°C using a high-pressure extruder under nitrogen protection to obtain DiR-labeled liposomes.

[0131] (1) Measurement of liposome particle size and zeta potential

[0132] 100 μL of liposome solution was diluted to 2 mL with physiological saline and mixed thoroughly. The particle size distribution, zeta potential, and polymer dispersity index (PDI) were analyzed using a laser scattering particle size analyzer (Malvern Zetasizer Nano ZS90, Worcestershire, UK). The results are shown in Table 1 below.

[0133] Table 1. Liposome parameters modified with different ligands

[0134]

[0135] The values ​​in the table are represented as: ( n=3)

[0136] As shown in the table above, the DiR-labeled liposomes prepared by the thin-film dispersion-high-pressure extrusion method are simple to prepare, have uniform particle size (PDI < 0.1), and the particle size is between 65-75 nm. The ZETA potential values ​​of the liposomes are positively charged, ranging from 8-33 mV.

[0137] (2) Liposome morphology characterization

[0138] The morphology of liposomes was observed using negative staining transmission electron microscopy. A sample was dropped onto a copper grid. After 2 minutes, excess liquid was blotted away from the grid edge with filter paper. Then, phosphotungstic acid (3%, pH 7.0) was added to the copper grid. After 2 minutes, excess staining solution was blotted away from the grid edge with filter paper. Finally, pure water was added to the copper grid, and excess water was blotted away from the grid edge with filter paper. After drying, the liposomes were ready for electron microscopy observation. See appendix for details. Figure 1 Transmission electron microscopy images show ( Figure 1 The liposomes were round with clear edges, and their particle size ranged from 60 to 80 nm, with a uniform particle size distribution, which was basically consistent with the results measured by laser scattering particle size analyzer. DOX loaded into the aqueous phase within the liposomes appeared as a white, coffee-bean-like gel, a result consistent with literature reports.

[0139] Example 10: Liposome Liver Targeting Assay

[0140] (1) HepG2 cell uptake experiment

[0141] The human hepatocellular carcinoma line HepG2 overexpresses ASGPr; therefore, HepG2 was used as a cell model to compare and contrast the uptake of DiR-labeled ligand-modified liposomes by HepG2 cells. Normally cultured HepG2 cells were digested, counted, and their density adjusted to 1x10⁶ cells / cells with serum-containing culture medium. 5 ·mL -1 1 mL of culture medium was seeded into each well of a 24-well plate, and 1 mL of culture medium was added to each well. The plates were then incubated in a 5% CO2 incubator for 24 hours. After removing the culture medium, DiR-labeled ligand-modified liposomes were added to each well, adjusting the DiR concentration to 0.2 g / mL. -1 Incubate at 37℃ in a 5% CO2 incubator for 1 hour, then remove the culture medium and wash three times with pre-cooled pH 7.4 PBS. Add 0.4 mL of trypsin to each well and digest at 1000 rpm. -1 Centrifuge for 5 min, discard the supernatant, add 1 mL of pH 7.4 PBS, resuspend, centrifuge, wash 3 times, add 500 μL of PBS to prepare a cell suspension, and analyze cell fluorescence intensity by flow cytometry. The experiment was repeated 3 times, and the results are shown in Table 2. Figure 2Using PBS-treated cells as a control group, the average fluorescence intensity of the single-antennae Gal ligand modified liposomes (DiR-LP-PEG2-6-GalNAc, DiR-LP-6-GalNAc) was significantly higher than that of the ordinary liposome group (DiR-Unmod-LP) (P<0.05), being 1.4 times and 6.4 times that of the DiR-Unmod-LP group, respectively. This may be related to the specific recognition of galactose modified on the liposome surface by ASGPr. The average fluorescence intensity of the lipid modified with dual-antenna Gal ligand (DiR-LP-6-(GalNAc)2) was significantly higher than that of the liposome modified with single-antenna Gal ligand (P<0.01), being 3.34 times that of DiR-LP-PEG2-6-GalNAc and 15.07 times that of DiR-LP-6-GalNAc, respectively. This indicates that the uptake efficiency of Gal ligand by liver cancer cells increases significantly with the increase of the number of antennae. This is because multi-antenna Gal ligands can occupy multiple ASGPr binding sites simultaneously, resulting in a much higher affinity than single-antenna Gal ligands, i.e., a clustering effect. (2) HepG2 cell uptake inhibition experiment

[0142] Normally cultured HepG2 cells were taken, digested, counted, and their density was adjusted to 1x10⁻⁶ cells using serum-containing culture medium. 5 ·mL -1 1 mL of culture medium was seeded into each well of a 24-well plate, and 1 mL of culture medium was added to each well. The plates were then incubated in a 5% CO2 incubator for 24 h. After removing the culture medium, 20 mM GalNAc was added and the plates were incubated for 30 min. Then, DiR-labeled ligands were added to modify the liposomes, and the DiR concentration was adjusted to 0.2 g / mL. -1 The subsequent operations are the same as in item (1). The results are shown in Table 2 and Figure 2 As shown.

[0143] Table 2. Cellular uptake of liposomes with different ligand modifications

[0144]

Claims

1. A liver-targeting glycoligand molecule, the general structural formula of which is as follows: in, R1=CH3CONH-, R2=CH2, R3=CH2; n=1-9.

2. The liver-targeting glycoligand molecule as described in claim 1, characterized in that, The n = 1-6.

3. The liver-targeting glycoligand molecule as described in claim 2, characterized in that, The structure is as follows:

4. A drug delivery system comprising a liver-targeting glycoligand molecule as described in any one of claims 1-3, the drug delivery system comprising:

5. The drug delivery system as described in claim 4, characterized in that, The drug delivery system includes: The drug delivery system also includes other pharmaceutically acceptable excipients.

6. The drug delivery system as described in claim 5, characterized in that, The drugs are selected from doxorubicin, epirubicin, paclitaxel, docetaxel, cisplatin, vincristine, lenvatinib, and siRNA drugs for treating liver diseases.

7. Use of a liver-targeting glycoligand molecule as described in any one of claims 1-3 or a drug delivery system as described in any one of claims 4-6 in the preparation of a medicament for treating liver diseases.

8. A method for preparing the liver-targeting glycoligand molecule as described in any one of claims 1-3, wherein the preparation method is carried out by bio-enzymatic catalysis, and the reaction route is as follows: in, The bioenzyme is selected from one or more of Novozym 435, PS IM, CRL, and TL IM; the reaction temperature is 10-70℃; the reaction solvent is an organic solvent selected from one or more of isooctane, tetrahydrofuran, n-hexane, acetone, tert-butanol, benzene, toluene, and pyridine, or a combination thereof.

9. The preparation method according to claim 8, characterized in that, The reaction temperature is 30-60℃.

10. The preparation method according to claim 8 or 9, characterized in that, The reaction route is as follows:

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