A halogenated benzocyclooctyne-polyethylene glycol modified halofuginone and a preparation method and application thereof

Abstract

The application discloses a method for hydrophilic modification of halenaquinone by carboxyl polyethylene glycol with a benzo cyclooctyne group to obtain benzo cyclooctyne-polyethylene glycol-halenaquinone (CPH) with good biocompatibility, and uses CPH for anti-tissue fibrosis treatment through glycometabolism engineering and orthogonal click chemistry. Compared with halenaquinone small molecules, CPH has negligible cytotoxicity. CPH has good therapeutic effect on tissue fibrosis in combination with glycometabolism engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological materials and medical technology, in particular to a halogenated furanone-polyethylene glycol modified halofuginone with good biological safety and a preparation method and application thereof. BACKGROUND

[0002] Tissue fibrosis is a disease caused by disease triggers or tissue damage, which leads to excessive deposition of connective tissue components in organs. Excessive accumulation of extracellular matrix (ECM) proteins often disrupts the physiological structure of the tissue and can cause organ dysfunction. Fibrotic tissue remodeling can affect almost all organ systems. Although the incidence of most individual fibrotic diseases is low, the fibrotic tissue response is very common in asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis or chronic inflammatory bowel disease. For example, myocardial remodeling in heart failure, epithelial-mesenchymal transition and profibrotic response in tumors, and airway remodeling in COPD and asthma. Common diseases associated with fibrosis include hepatitis virus infection, non-alcoholic fatty liver disease (NAFLD), chronic kidney disease, idiopathic pulmonary fibrosis (IPF), myocardial infarction, systemic sclerosis, pneumoconiosis and cystic fibrosis. The annual combined incidence of major fibrosis-related diseases is about 4968 / 100000 people, causing a huge disease burden. Therefore, tissue fibrosis is increasingly recognized as a major health challenge.

[0003] Halofuginone (HF) was first synthesized in the 1960s as a potential antimalarial drug, which is a derivative of halofuginone, an active ingredient of traditional Chinese medicine, and is a competitive prolyl-tRNA synthetase. In recent years, some researchers have reported that HF can degrade the immunosuppressive extracellular matrix of pancreatic ductal adenocarcinoma; it can also reduce osteoarthritis by inhibiting the activity of TGFβ. Halofuginone is also an effective pulmonary vasodilator that can activate Kv channels and block voltage-gated calcium channels. Halofuginone also has good anti-inflammatory and tumor cell-killing effects. Although halofuginone has antimalarial, extracellular matrix-degrading, anti-inflammatory and vasodilating effects, halofuginone itself is highly toxic to cells, with an IC50 value in the nanogram range, so its application in vivo is very limited. It is worth noting that the application of halofuginone in the treatment of tissue fibrosis has not been systematically studied, and more importantly, the mechanism of halofuginone in anti-fibrosis is still unclear. Therefore, it is very important to better utilize the anti-fibrosis effect of halofuginone in the treatment of tissue fibrosis while ensuring biological safety. SUMMARY

[0004] The present application is directed to the deficiencies of the prior art, and synthesizes a benzocyclooctyne polyethylene glycol modified halofuginone (CPH), which utilizes the biocompatible polyethylene glycol macromolecule to reduce the biological toxicity of halofuginone. By glycometabolism engineering, an azido group is expressed in fibrotic tissue, and the click chemistry reaction of the azido group and the cycloalkyne group is used to actively target the treatment effect of CPH on fibrosis.

[0005] The present application aims to provide a CPH with good biosafety and combine glycometabolism engineering technology to improve the anti-fibrosis effect of CPH.

[0006] The technical solution of the present application is as follows:

[0007] A benzocyclooctyne-polyethylene glycol modified halofuginone has the following structure:

[0008]

[0009] Wherein: n = 10-110.

[0010] The above-mentioned polyethylene glycol modified halofuginone is prepared by room temperature condensation reaction of carboxyl polyethylene glycol (DBCO-PEG-COOH) with a benzocyclooctyne group and the hydroxyl group of halofuginone.

[0011] The PEG used in the present application can be PEG500, PEG1000, PEG2000, PEG5000.

[0012] The halofuginone (HF) has the following structure:

[0013]

[0014] Another object of the present application is to provide a method for preparing the above-mentioned benzocyclooctyne-polyethylene glycol modified halofuginone, comprising the following steps:

[0015] Step one: dissolve DBCO-PEG-COOH and halofuginone with different molecular weights in an aprotic solvent, then add an appropriate amount of N,N'-diisopropyl carbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) for reaction.

[0016] The above-mentioned method preferably reacts at room temperature for 36 hours.

[0017] Preferably, the molar ratio of DBCO-PEG-COOH: halofuginone: DIC: DMAP is 1:1.2:1.5:0.5.

[0018] Preferably, the aprotic solvent is selected from N,N dimethylformamide and / or formamide. Preferably, it is N,N dimethylformamide.

[0019] The method further comprises the steps of removing the reaction solvent and drying.

[0020] Another object of the present application is to provide the use of the benzocyclooctyne-polyethylene glycol modified halofuginol in the treatment of tissue fibrosis.

[0021] The benzocyclooctyne-polyethylene glycol modified halofuginol is used in combination with sugar metabolism engineering, and an azido group is expressed by a sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine at a fibrosis site of a patient, and then a click chemistry reaction of the azido group and the benzocyclooctyne-polyethylene glycol modified halofuginol is used to specifically enrich the benzocyclooctyne-polyethylene glycol modified halofuginol at the lesion site.

[0022] The sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine has the following structure: In one specific example of the present application, CPH combined with sugar metabolism engineering can be used to treat liver fibrosis.

[0023] Advantages of the present application:

[0024] (1) The benzocyclooctyne-polyethylene glycol modified halofuginol of the present application can be used in combination with sugar metabolism engineering to increase the accumulation and residence time of halofuginol at the lesion site, and can better treat tissue fibrosis.

[0025] (2) The preparation method of CPH is simple, and CPH has good biological safety compared with small molecule halofuginol. BRIEF DESCRIPTION OF DRAWINGS

[0026] The present application is further described below in combination with the drawings and examples.

[0027] Figure 1 is the nuclear magnetic characterization diagram of CPH.

[0028] Figure 2 is the cytotoxicity of HF on hepatic stellate cells LX2.

[0029] Figure 3 is the cytotoxicity of CPH on hepatic stellate cells LX2.

[0030] Figure 4 is the fluorescence imaging of the sugar metabolism engineering marker of hepatic stellate cells LX2.

[0031] Figure 5 is the treatment of liver fibrosis by CPH combined with sugar metabolism engineering. DETAILED DESCRIPTION

[0032] The content of the present application is further illustrated below in combination with examples, but these examples do not limit the protection scope of the present application.

[0033] Example 1: Preparation of benzocyclooctyn-polyethylene glycol-crystalone (CPH)

[0034] Commercial DBCO-PEG 5000 -COOH (Shanghai Pengshuo Biotechnology Co., Ltd.) and styraxone were dissolved in N,N-dimethylformamide (DMF) at a molar ratio of 1:1.2. Then, appropriate amounts of N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) were added to the reaction solution, and the mixture was stirred overnight at room temperature. The ratio of DBCO-PEG-COOH:styraxone:DIC:DMAP was 1:1.2:1.5:0.5. After the reaction was completed, the DMF was evaporated to dryness using a vacuum rotary evaporator. The solid was dissolved in water, and the solution was filtered through a 220 μm water-soluble filter membrane. The treated aqueous solution was then lyophilized.

[0035] Structural characterization data of the product, such as Figure 1 As shown, letter 'a' represents the characteristic peak of the DBCO group, with a chemical shift of 7.2-7.8 ppm; letter 'b' represents the characteristic peak of the polyethylene glycol backbone, with a chemical shift of 3.5 ppm indicating the characteristic peak of a repeating unit and a chemical shift of 4.09 ppm indicating the characteristic peak of the methylene group near the carboxyl group; letter 'c' represents the characteristic peak of halogenone. The linkage efficiency of halogenone can be calculated to be 60% by integrating the characteristic peak areas.

[0036] Its structure is as follows:

[0037]

[0038] Example 2: CPH Cytotoxicity Assay

[0039] Hepatic stellate cells (LX2) in culture flasks were digested and seeded into 96-well plates at a density of 5000 cells / well. Cells were cultured overnight until adherence. The next day, cells were co-cultured with different concentrations of CPH and fentanyl sulfoxide (HF) for 48 hours. The CPH concentration was based on an equivalent volume of HF (HF concentrations of 10, 20, 40, 60, 80, and 100 nM). After 48 hours, the old culture medium was aspirated, and 180 μL of fresh culture medium and 20 μL of MTT solution (5 mg / mL) were added. The cells were incubated for another 4 hours. The liquid in each well was aspirated, and 150 μL of DMSO was added to dissolve the cells in the dark for 10 minutes. The absorbance of each well at 562 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (Absorbance of test group - Background absorbance) / (Absorbance of control group - Background absorbance) × 100%.

[0040] The results are as follows Figure 2 and Figure 3As shown, HF is highly toxic to cells, while the modified CPH can significantly downregulate the toxicity of HF and has good biocompatibility.

[0041] Example 3: Glycolysis Engineering to Express Azide on Cell Surface

[0042] After digestion, hepatic stellate cells (LX2) were processed at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 35 mm in small dishes with slides. Cells were cultured overnight in 2 mL of DMEM medium, followed by 48 hours of incubation with 25 μM Ac4ManNAz. After washing three times with PBS, cells were added to 2 mL of fresh DMEM medium, followed by 5 μM DBCO-Cy5, and incubated for 1 hour. After incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature in the dark for 10 minutes, washed three times with PBS, stained with DAPI at room temperature in the dark for 10 minutes, washed three times with PBS again, and observed under a 63x oil immersion microscope using a laser confocal microscope.

[0043] The results are as follows Figure 4 As shown, glycogen metabolism engineering can successfully express azide groups on the surface of LX2 cells.

[0044] Example 4: CPH and glucose metabolism engineering for the treatment of liver fibrosis

[0045] Mice with liver fibrosis were injected via tail vein with 20 mg / kg Ac4ManNAz or PBS once daily for three days. Three days later, the mice were injected via tail vein with CPH (0.5 mg / kg HF) or PBS every two days for one week. Healthy ICR mice served as negative controls. One week after the completion of all drug administration, the mice were sacrificed, major organs were removed, and liver tissue was frozen and sectioned. The liver sections were stained with type I collagen, and the immunofluorescence of the sections was observed using a laser confocal microscope after staining.

[0046] The results are as follows Figure 5 As shown, the combination of CPH and glucose metabolism engineering can effectively treat liver fibrosis.

Claims

1. A benzocyclooctyne-polyethylene glycol modified halenaquinone, characterized by having the following structure: wherein: n = 10-110.

2. A process for the preparation of the benzocyclooctyne- polyethylene glycol modified halofuginolides of claim 1, characterized by, The condensation reaction is prepared by the carboxyl polyethylene glycol DBCO-PEG-COOH with benzocyclooctyne group and the hydroxyl of halenaquinone.

3. The method of claim 2, wherein DBCO-PEG-COOH and halenaquinone are dissolved in an aprotic solvent, and N,N'-diisopropyl carbodiimide and 4-dimethylaminopyridine are added to carry out condensation reaction.

4. The method of claim 3, wherein The reaction temperature is room temperature, and the reaction time is 36 hours.

5. The method of claim 3, wherein The aprotic solvent is selected from N,N dimethylformamide and / or formamide.

6. The method of claim 3, wherein The molar ratio of DBCO-PEG-COOH: halenaquinone: N,N'-diisopropyl carbodiimide: 4-dimethylaminopyridine is = 1:1.2:1.5:0.

5.

7. The method of claim 3, wherein The PEG in DBCO-PEG-COOH is PEG500, PEG1000, PEG2000 or PEG5000.

8. The use of the benzocyclooctyne-polyethylene glycol modified halenaquinone of claim 1 in the preparation of a medicament for treating tissue fibrosis.

9. Use according to claim 8, characterized in that The benzocyclooctyne-polyethylene glycol modified halenaquinone is used in combination with sugar metabolism engineering, and the azido group is expressed by sugar metabolism engineering at the fibrosis site of the patient from the sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine, and then the click chemistry reaction of the azido group and the benzocyclooctyne-polyethylene glycol modified halenaquinone is used to specifically enrich the benzocyclooctyne-polyethylene glycol modified halenaquinone at the lesion site.

10. Use according to claim 8, characterized in that The tissue fibrosis is liver fibrosis.

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