A glycosyl gallic acid derivative and its preparation method and application in the preparation of targeted diagnosis and treatment materials for liver diseases

Abstract

The present invention discloses a glycosyl gallic acid derivative, the general structural formula of which is shown below: The present invention designs and synthesizes a class of glycosyl-modified gallic acid derivatives with three-branched benzoic acid as a characteristic mother nucleus, wherein two sugar-type molecules, acetylgalactosamine and galactose, are modified with the three-branched benzoic acid as the characteristic mother nucleus. These are modified on the surface of drugs or drug delivery systems, thereby realizing targeted diagnosis and treatment of liver diseases, overcoming the defect of low affinity for targeting cancer cells in the prior art.

Description

Technical Field

[0001] The present invention belongs to the technical field of diagnosis and treatment of liver diseases. Specifically, it relates to a glycosyl-modified gallic acid derivative that can be recognized and bound to the asialoglycoprotein receptor (ASGPR) on the surface of liver cancer cells, as well as its preparation method and use in constructing targeted diagnosis and treatment materials for liver diseases. Background Art

[0002] Liver cancer, hyperlipidemia, etc. are common liver diseases in humans. Their treatment methods include external medication, surgery, chemotherapy, immunotherapy, targeted therapy, etc. Among them, glycosylation-modified drug delivery systems are an active targeted therapy for the diagnosis and treatment of liver diseases. They can overcome the problems of wide distribution of drugs in the body and serious toxic side effects. At present, the commonly used active targeting glycoside ligands mainly include galactose, acetylgalactosamine, etc. Studies have found that drug delivery systems modified with acetylgalactosamine and galactose can regulate their biodistribution and induce specific uptake of liver cells through the mediation of asialoglycoprotein receptors, significantly improving the accumulation of drugs in the liver, and have great application value in the medical field. Direct glycosylation modification of drugs can improve their targeting to specific organs. Glycosylation modification of drug carriers can deliver target drugs, antigens and nucleic acids to specific cells, exerting the targeted delivery function of drug delivery systems.

[0003] Based on the advantages of the above-mentioned glycosylation modification, we designed and synthesized trivalent Gal, GalNAc oligosaccharides with high affinity for ASGPR receptors, and modified them into drug delivery systems in combination with nanolipid delivery systems to prepare siRNA cationic liposomes with targeted effects. Our research investigated their biological activities in vivo and in vitro, laying the foundation for preclinical research on targeted small interfering RNA molecular drugs, thereby achieving specific and precise diagnosis and treatment of related diseases. Summary of the Invention

[0004] The first object of the present invention is to provide a glycosyl gallic acid derivative.

[0005] The second object of the present invention is to provide a method for preparing the glycosyl gallic acid derivatives.

[0006] The third object of the present invention is to provide an application of the glycosyl gallic acid derivatives in the preparation of targeted diagnostic and therapeutic materials for liver diseases.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a glycosyl gallic acid derivative, the general structural formula of which is shown below:

[0009]

[0010] Wherein, m is a positive integer selected from 1 to 5, preferably 1, 2, or 3;

[0011] n is a positive integer selected from 2 to 6, preferably 2, 3, 4, 5, 6;

[0012] q is a positive integer selected from 2 to 7, preferably 2, 3, 4, 5, 6, 7;

[0013] r is a positive integer selected from 2 to 8, preferably 2, 3, 4, 5, 6, 7, 8;

[0014] R1 is selected from Preferably, the structure of the glycosyl gallic acid derivative is selected from one of the following structures:

[0015]

[0016]

[0017] The second aspect of the present invention provides a method for preparing the glycosyl gallic acid derivatives, comprising the following steps:

[0018]

[0019] In the first step, methyl 3,4,5-trihydroxybenzoate, tert-butyl (2-bromoethyl)carbamate, and Cs2CO3 in a molar ratio of 1:(2-6):(3-10) are dissolved in acetonitrile, and the mixture is heated to 75-85°C and refluxed with stirring to obtain a compound represented by formula A.

[0020]

[0021] In the second step, the compound represented by formula A and sodium hydroxide at a molar ratio of (0.01-0.5):1 are dissolved in methanol and reacted overnight to obtain a crude product;

[0022] The crude product, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to dichloromethane, activated in an ice bath for 1-14 hours, propargylamine and triethylamine are added in a molar ratio of the crude product, propargylamine, and triethylamine of 1:1:(1-4), and reacted overnight in an ice bath to obtain a compound represented by formula B;

[0023]

[0024]

[0025] In the third step, the compound represented by formula B was added to DCM and TFA in a volume ratio of 1:1, and the mixture was stirred under ice bath conditions to obtain a crude product, compound C;

[0026] N-Boc-γ-aminobutyric acid, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to DMF, activated for 1-12 hours under ice bath conditions, compound C and triethylamine are added, and the molar ratio of N-Boc-γ-aminobutyric acid, compound C, and triethylamine is (1-4):1:(10-50), and the reaction is carried out under ice bath conditions overnight to obtain a compound represented by formula D;

[0027]

[0028]

[0029] Step 4: Add the compound represented by Formula D to DCM and TFA in a volume ratio of 1:1, and stir the mixture under ice bath conditions to obtain a crude product, Compound E;

[0030] Compound F, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to DMF, activated in an ice bath for 1-12 hours, and compound E and triethylamine are added in a molar ratio of (1-4):1:(10-50) to obtain a compound represented by formula G.

[0031] Compound G is added to an excess of methanol / sodium methoxide solution, stirred at room temperature for 1 to 4 hours, the reaction is stopped, a cation exchange resin is added, the pH is adjusted to neutral, filtered, and distilled under reduced pressure to obtain a colorless oil to obtain the compound represented by Formula IA;

[0032] Alternatively, compound F is replaced by the following structure:

[0033]

[0034] Compound IB was prepared;

[0035]

[0036] The third aspect of the present invention provides a use of the glycosyl gallic acid derivatives in the preparation of targeted diagnostic and therapeutic materials for liver diseases.

[0037] The liver disease is liver cancer.

[0038] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0039] The present invention designs and synthesizes a class of glycosyl-modified gallic acid derivatives with three-branched benzoic acid as the characteristic mother core. Two sugar-type molecules, acetylgalactosamine and galactose, are modified with three-branched benzoic acid as the characteristic mother core. These molecules are modified on the surface of drugs or drug delivery systems to achieve targeted diagnosis and treatment of liver diseases, overcoming the defect of low affinity for targeting cancer cells in the existing technology.

[0040] The glycosyl-modified gallic acid derivatives of the present invention increase their binding affinity to the corresponding glycosyl receptor ASGPR by multivalently modifying monovalent glycosyl ligands and utilizing the antennae structure formed by natural oligosaccharides. By varying the size of n, the glycosyl-modified gallic acid derivatives can be modified on the surfaces of various material structures, such as liposomes, a drug delivery system, to construct a lipid drug active targeted delivery system. By varying the R1 group, specific targeting of different glycosyl receptors on the surfaces of various cancer cells can be achieved. Furthermore, altering the length of the linker between R1 and the benzene ring can improve and enhance receptor binding affinity, thereby increasing the targeting ability of the glycosyl ligand. This compound provides a rapid and simple new tool for the clinical diagnosis and treatment of various types of liver diseases.

[0041] The preparation method of the present invention is achieved through 9 to 11 reaction steps, with simple process conditions and post-processing, making it easy to implement. Firstly, the raw materials used in the present invention are inexpensive and readily available, and the reaction steps are simple, involving only bromination, amidation, and deprotection reactions. All reaction steps require simple post-processing, requiring only conventional separation methods such as extraction and column chromatography. Furthermore, with the exception of a few individual reaction steps, the yield of the remaining reaction products is consistently above 50%, representing a good yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the cell activity test results of Compound IA and Compound IB prepared in Example 1 and Example 2. DETAILED DESCRIPTION

[0043] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0044] Example 1

[0045] (1) Synthesis of the Core Compound of 3,4,5-Tris(2-((tert-butoxycarbonyl)amino)ethoxy)benzoic Acid Methyl Ester (Compound Represented by Formula A)

[0046]

[0047] To a 100 mL round-bottom flask, methyl 3,4,5-trihydroxybenzoate (700 mg, 3.80 mmol), tert-butyl (2-bromoethyl)carbamate (2.56 g, 11.49 mmol), and Cs2CO3 (8.67 g, 26.6 mmol) were added. The mixture was heated to 80°C in 50 mL of MeCN and stirred under reflux. After 12 hours of reaction, TLC indicated completion of the reaction, and the reaction was terminated. The product was evaporated under reduced pressure, filtered, and concentrated. Column chromatography (PE:EA = 1:1, v / v) afforded 2 g of the desired compound (Formula A) as a white solid.

[0048] (2) Synthesis of the compound represented by formula B

[0049]

[0050] Compound A (500 mg, 0.82 mmol) was weighed and placed in a 250 mL round-bottom flask. 10 mL of 1M NaOH solution and 20 mL of methanol were added. After overnight reaction, TLC monitoring indicated complete reaction of Compound A. HCl was added to adjust the pH to 4. The mixture was extracted with CHCl, dried over anhydrous NaSO, and filtered to obtain the crude product.

[0051] The crude product (200 mg, 0.33 mmol), EDC·HCl (76 mg, 0.40 mmol), and HOBt (77 mg, 0.40 mmol) were weighed and added to 15 mL of dichloromethane. The mixture was activated on ice for 10 h. Propargylamine (18 mg, 0.33 mmol) and triethylamine (94 mg, 0.92 mmol) were added and the mixture was allowed to react overnight on ice. TLC indicated completion of the reaction. The reaction was stopped and the solvent was evaporated to give the crude product. The product was separated by column chromatography (DCM:MeOH = 20:1, v / v) to afford the compound represented by Formula B.

[0052] (3) Synthesis of the compound represented by formula D

[0053]

[0054] Compound B (50 mg, 0.079 mmol) was weighed and added to 10 mL of DCM / TFA (v / v = 1:1). The mixture was stirred on ice for 2 h to remove the Boc protection. TLC indicated that the reaction of Compound B was complete. The reaction was stopped and the crude product, Compound C, was obtained by distillation under reduced pressure.

[0055] N-Boc-γ-aminobutyric acid (43 mg, 0.21 mmol), EDC·HCl (50 mg, 0.26 mmol), and HOBt (34 mg, 0.26 mmol) were weighed and added to 10 mL of DMF. The mixture was activated on ice for 10 h. Compound C (35 mg, 0.071 mmol) and triethylamine (178 mg, 1.76 mmol) were added and allowed to react overnight on ice. TLC indicated completion of the reaction. The reaction was stopped and the solvent was evaporated to give the crude product. The product was separated by column chromatography (DCM:MeOH = 20:1, v / v) to afford the compound represented by Formula D.

[0056] (4) Synthesis of the compound represented by formula IA

[0057]

[0058] Compound D (50 mg, 0.056 mmol) was weighed and added to 10 mL of DCM / TFA (v / v = 1:1). The mixture was stirred on ice for 2 h to remove the Boc protection. TLC indicated that the reaction of Compound D was complete. The reaction was stopped and the crude product, Compound E, was obtained by distillation under reduced pressure.

[0059] Compound F (100 mg, 0.22 mmol), EDC·HCl (50 mg, 0.26 mmol), and HOBt (34 mg, 0.26 mmol) were weighed and added to 10 mL of DMF. The mixture was activated on ice for 10 h. Compound E (44 mg, 0.074 mmol) and triethylamine (178 mg, 1.76 mmol) were added and allowed to react overnight on ice. TLC indicated completion of the reaction. The reaction was stopped and the solvent was evaporated to give the crude product. The product was separated by column chromatography (DCM:MeOH = 20:1, v / v) to afford the compound represented by Formula G.

[0060] Compound G (20 mg, 0.010 mmol) was weighed and added to a 10 mL methanol / sodium methoxide solution (10 mg sodium methoxide dissolved in 10 mL methanol). The mixture was stirred at room temperature for 2 h. TLC indicated completion of the reaction, and the reaction was stopped. A cation exchange resin was added, the pH was adjusted to neutral, and the mixture was filtered and distilled under reduced pressure to obtain a colorless oil, the compound of Formula IA. 1HNMR(400MHz,MeOD)δ7.16(s,1H),4.18(dd,J=7.2,1.6Hz,1H),4.09(dt,J=18.3, 5.5Hz,3H),3.88(ddd,J=9.1,7.2,5.4Hz,2H),3.80(d,J=3.0Hz,1H),3.74–3.67(m ,3H),3.66–3.56(m,8H),3.56–3.43(m,5H),3.17(td,J=7.0,3.4Hz,3H),2.30–2.1 5(m,7H),1.77(h,J=7.1,6.6Hz,2H),1.66(q,J=7.3Hz,2H),1.58(t,J=7.2Hz,3H).

[0061] Example 2

[0062] The compound F in step (4) of Example 1 can be replaced with carboxyacetylgalactosamine as shown below,

[0063]

[0064] Other raw materials, reagents and steps used are similar to those in Example 1 to obtain the compound represented by Formula IB. 1 HNMR(400MHz,MeOD)δ7.11(s,2H),5.34(d,J=3.3Hz,3H),5.19(d,J=11.0Hz,3H),4.57(t,J=7.9Hz, 3H),4.18–4.05(m,15H),3.68–3.60(m,17H),3.48(s,18H),2.33–2.21(m,12H),1.70–1.49(m,18H).

[0065]

[0066] Example 3

[0067] Compound IA and Compound IB prepared in Example 1 and Example 2 were subjected to cell activity test. Test method: HepG2 cells were seeded into 96-well plates at a rate of 25,000 cells per well and treated with different concentrations of Compound IA and Compound IB prepared in Example 1 and Example 2 for 24 hours. CCK-8 reagent was used to incubate the cells for 40 minutes, and the absorbance of the cells in the 96-well plates at 450 nm was measured using a multifunctional microplate reader. The results are shown in FIG. Figure 1 As shown, Figure 11 is a schematic diagram of the cell activity test results of Compound IA and Compound IB prepared in Example 1 and Example 2. The results show that both can produce a lethality rate of nearly 50% on liver cancer HepG2 cells at a concentration of 20 nM, verifying their targeting effect on liver cancer cells.

[0068] Example 4

[0069] Galactose is a monosaccharide widely found in dairy products and sugar beets and is an important component of certain glycoproteins. There is a type of galactose receptor on the surface of liver cells, also known as the Asialoglycoprotein receptor (ASGPR). ASGPR is an ideal active targeting receptor: it is highly expressed on liver cells but not on other cell types, and each cell contains (1 to 5)*10 5 The binding site for GalNAc is located in the cell surface. This leads to rapid internalization of GalNAc after binding, followed by rapid recycling to the cell surface. Receptor-mediated endocytosis allows for the uptake of large quantities of Gal and GalNAc for catabolism and recycling. Therefore, acetylgalactosamine and galactose can serve as targeting modifiers to achieve targeted drug delivery to liver cancer cells.

[0070] Liposome is the lipid vesicle that is made up of phospholipid molecules, and because of its mature industrial manufacturing technology, high cell absorption, low cytotoxicity and low immunogenicity, it becomes one of the most successful delivery systems. As a drug delivery carrier, it has sustained-release drugs, prolongs drug action time, can be delivered to the diseased part by modifying the targeting molecule, reduces drug dosage, avoids drug resistance, reduces the advantages such as the damage to normal tissue. In addition, its surface can also modify various targeting ligands, such as antibodies, peptides and carbohydrates. The modification of ligand can make liposome selectively target the diseased cells that overexpress specific cell surface receptors, and the compound IA and compound IB prepared by the embodiment of the present invention 1 and embodiment 2 can specifically target the asialoglycoprotein receptor (ASGPR) that overexpresses on the surface of liver cancer cells. This active targeting can significantly increase the dosage, improves efficacy and reduces side effects.

[0071] Appropriate modification of galactose or acetylgalactosamine ligands on lipid nanomaterial delivery carriers can be used to specifically target liver cells. Once attached to the surface of liver cells, the siRNA-containing complex can enter the cell through receptor-mediated endocytosis. Compound IA and Compound IB prepared in Examples 1 and 2 of the present invention are designed with alkynyl groups, such as dibenzocyclooctyne, terminal alkynes, etc., and the lipid components are modified with azide groups. By SPAAC or CuAAC reactions, glycosyl ligands can be bound to the liposome surface, thereby obtaining targeted liposomes, specifically identifying liver cells that specifically express ASGPR receptors such as liver cancer cells, and achieving targeted diagnosis and treatment of liver diseases. Compound IA and Compound IB prepared in Examples 1 and 2 of the present invention can be used as targeted diagnosis and treatment materials for liver diseases.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A glycosyl gallic acid derivative, characterized in that: The general structural formula is shown below: wherein m is a positive integer selected from 1 to 5; n is a positive integer selected from 2 to 6; q is a positive integer selected from 2 to 7; r is a positive integer selected from 2 to 8; R1 is selected from 2. The glycosyl gallic acid derivative according to claim 1, characterized in that: The structure of the glycosyl gallic acid derivative is selected from one of the following structures:

3. A method for preparing the glycosyl gallic acid derivatives according to claim 1 or 2, characterized in that: The following steps are involved: In the first step, methyl 3,4,5-trihydroxybenzoate, tert-butyl (2-bromoethyl)carbamate, and Cs2CO3 in a molar ratio of 1:(2-6):(3-10) are dissolved in acetonitrile, and the mixture is heated to 75-85°C and refluxed with stirring to obtain a compound represented by formula A. In the second step, the compound represented by formula A and sodium hydroxide at a molar ratio of (0.01-0.5):1 are dissolved in methanol and reacted overnight to obtain a crude product; The crude product, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to dichloromethane, activated in an ice bath for 1-14 hours, propargylamine and triethylamine are added in a molar ratio of the crude product, propargylamine, and triethylamine of 1:1:(1-4), and reacted overnight in an ice bath to obtain a compound represented by formula B; In the third step, the compound represented by formula B was added to DCM and TFA in a volume ratio of 1:1, and the mixture was stirred under ice bath conditions to obtain a crude product, compound C; N-Boc-γ-aminobutyric acid, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to DMF, activated for 1-12 hours under ice bath conditions, compound C and triethylamine are added, and the molar ratio of N-Boc-γ-aminobutyric acid, compound C, and triethylamine is (1-4):1:(10-50), and the reaction is carried out under ice bath conditions overnight to obtain a compound represented by formula D; Step 4: Add the compound represented by Formula D to DCM and TFA in a volume ratio of 1:1, and stir the mixture under ice bath conditions to obtain a crude product, Compound E; Compound F, EDC·HCl, and HOBt in a molar ratio of 1:(1-2):(1-2) are added to DMF, activated in an ice bath for 1-12 hours, and compound E and triethylamine are added in a molar ratio of (1-4):1:(10-50) to obtain a compound represented by formula G. Compound G is added to an excess of methanol / sodium methoxide solution, stirred at room temperature for 1 to 4 hours, the reaction is stopped, a cation exchange resin is added, the pH is adjusted to neutral, filtered, and distilled under reduced pressure to obtain a colorless oil to obtain the compound represented by Formula IA; Alternatively, compound F is replaced by the following structure: Compound IB was prepared; 4. Use of the glycosyl gallic acid derivatives according to claim 1 or 2 in the preparation of targeted diagnostic and therapeutic materials for liver diseases.

5. The use according to claim 4, characterized in that The liver disease is liver cancer.

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