Water-soluble fullerenes and intermediates thereof, methods of preparation and uses

Abstract

The present application relates to a kind of water-soluble fullerene and intermediate, preparation method and application, it is related to material field, including for the compound of hydrophilic treatment to fullerene, and including its water-soluble fullerene, water-soluble fullerene it includes fullerene and the polyhedral molecular cage that it is wrapped, the polyhedral molecular cage is formed by compound M1 (6-vinyl pyridyl hexaphenylbenzene) self-assembly.The present application is assembled by the hydrophobic effect of six molecules in water, forms a water-soluble C 60 Molecular perfectly wrapped in cavity cubic supramolecular cage (6M1@C 60 ).It significantly improves the water-solubility of C 60 Without changing the original structure of C 60 .

Description

Technical Field

[0001] The present invention relates to the field of materials, and in particular to a water-soluble fullerene and an intermediate thereof, a preparation method and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is the leading cause of death from cardiovascular-related diseases. Due to coronary artery obstruction, the myocardium suffers from insufficient blood supply, causing myocardial tissue hypoxia, leading to myocardial necrosis, fibrosis, and ventricular remodeling, seriously affecting cardiac function. In clinical practice, emergency PCI treatment, also known as reperfusion therapy, is the main method to alleviate AMI, because it can promptly relieve myocardial cell hypoxia, improve cardiac function, reduce the progression of infarct area, and effectively and promptly reduce mortality. However, during the ischemia-reperfusion process, reactive oxygen species (ROS) are produced in an explosive manner. When excessive, they may further lead to myocardial cell death, induce an aggravated inflammatory response, and even worse, cause myocardial contractile dysfunction, trigger malignant arrhythmias, and even endanger life.

[0003] The generation of reactive oxygen species (ROS) is based on sustained oxidative stress. The oxidative stress process has been shown to be involved in the pathogenesis of a variety of cardiovascular diseases, such as hypertension, aortic aneurysm, hypercholesterolemia, atherosclerosis, cardiac ischemia-reperfusion injury, myocardial infarction, heart failure and arrhythmia. Therefore, balancing the relationship between ROS and antioxidants is crucial for the normal function of cells. Current antioxidant treatment strategies mainly include dietary assistance, gene therapy, free radical scavengers, polyethylene glycol (PEG) conjugation and nanomedicine-based technologies. However, the effects and applications are not satisfactory. In view of the limitations of the above methods, there is an urgent need to seek an effective and powerful antioxidant to combat the development of the disease. Therefore, fullerenes with a strong ability to adsorb oxygen free radicals have become a possible application.

[0004] Fullerene, a "free radical sponge", has a unique electronic structure, high hardness, good stability, and magnetic and superconducting properties, which make it have broad application prospects in the fields of optics, catalysis, biomedicine, etc. However, due to the spherical shape and hydrophobic surface of fullerene, its solubility in water is limited, which greatly restricts its application. Most attempts are to chemically modify it, such as in C 60 Hydrophilic groups are introduced into the functionalized C-type cations, but functionalization will bring about certain performance losses, especially electron deficiency. 60 In the modification reaction, the introduction of double bonds will affect the C 60 The overall conjugation of C 60 Under the condition of the original structure, the ideal goal is to significantly improve its water solubility. Summary of the Invention

[0005] Technical issues

[0006] In view of this, the technical problem to be solved by the present invention is how to provide a water-soluble fullerene and its intermediates, preparation method and application.

[0007] Inspired by the traditional Lubansuo puzzle game, the present invention designs a hexagonal amphiphile (M1) that assembles into a water-soluble C-terminal through the hydrophobic interaction of six molecules in water. 60 The molecules are perfectly packed in the cubic supramolecular cage (6M1@C 60 ). It does not change C 60 Under the original structure, C 60 The water-soluble complex was further used to reduce ROS in cardiomyocytes (FMC84) through the Akt / Nrf2 / HO-1 pathway. 60 The construction of C 60 The important role of water-soluble fullerene in preventing oxidative stress-related cardiovascular disease damage was demonstrated, and the successful preparation of new water-soluble fullerenes and their feasibility and effectiveness in the medical field were confirmed.

[0008] Solution

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a compound for hydrophilizing fullerene, the structural formula of which is shown as (M1):

[0011]

[0012] In the second aspect, an intermediate of the compound described in the first aspect is provided, which is 6-vinylpyridylhexaphenylbenzene, and has the structural formula shown in (2).

[0013]

[0014] In a third aspect, a method for preparing the intermediate according to the second aspect is provided, comprising the following steps:

[0015] Under an inert gas atmosphere, hexabromophenylbenzene and p-vinylpyridine react in a solvent at 120-180° C. in the presence of a weak base salt and a catalyst to obtain a product of formula (2).

[0016] Furthermore, the weak base salt is one or more of sodium carbonate, potassium carbonate and cesium carbonate.

[0017] Optionally, the catalyst is PdCl2(PPh3)2 (bistriphenylphosphine palladium dichloride).

[0018] Optionally, the molar ratio of hexabromophenylbenzene to p-vinylpyridine is 1:(6-24), optionally 1:(8-16), optionally 1:(10-14), optionally 1:12.

[0019] Optionally, the molar ratio of vinyl pyridine to weak base salt is 1:(0.8-1.4), optionally 1:(1.0-1.2), optionally 1:1.

[0020] Optionally, the molar ratio of catalyst to hexabromophenylbenzene is (0.1-0.24):1, optionally 0.2:1.

[0021] Optionally, the solvent is a polar solvent, and optionally the solvent is anhydrous N,N-dimethylformamide.

[0022] Optionally, the reaction temperature is 130-180° C., optionally 140-160° C., optionally 150° C.; optionally, the reaction time is 40-60 h, optionally 48 h.

[0023] In a fourth aspect, a preparation method according to the first aspect is provided, wherein the intermediate of formula (2) according to the second aspect or the intermediate of formula (2) prepared by the preparation method according to the third aspect is reacted with iodomethane in a solvent under inert gas conditions to generate a product of formula (M1).

[0024] Furthermore, the molar ratio of the intermediate of formula (2) to methyl iodide is 1:(6-60), optionally 1:(12-54), optionally 1:(30-54), optionally 1:(40-54), optionally 1:(45-54), optionally 1:50.

[0025] and / or, the reaction temperature with methyl iodide is 35 to 60° C., optionally 40 to 60° C., optionally 40 to 50° C., optionally 45° C., optionally, the reaction time is 20 to 36 hours, optionally 24 hours;

[0026] Optionally, the solvent is a methyl halide, optionally chloroform.

[0027] In a fifth aspect, a water-soluble fullerene is provided, comprising a fullerene and a polyhedral molecular cage encapsulating the fullerene, wherein the polyhedral molecular cage is self-assembled by the compound M1 described in the first aspect or the compound M1 prepared by the preparation method of the fourth aspect.

[0028] Furthermore, the polyhedral molecular cage is self-assembled by 6 compounds M1.

[0029] And / or, the solubility of the water-soluble fullerene is 12 mg / ml.

[0030] And / or, the fullerene is C m , m is an integer between 30 and 90, optionally the fullerene is C 60 、C 70 One or more of the .

[0031] Furthermore, the polyhedral molecular cage has a hydrophobic cavity with a diameter of 1 to 2 nm.

[0032] In a sixth aspect, a method for preparing water-soluble fullerene is provided, comprising the following steps:

[0033] 1) heating a saturated aqueous solution of the compound M1 described in the first aspect or the compound M1 prepared by the preparation method of the fourth aspect at 80° C. to 100° C.;

[0034] 2) adding 6 to 12 equivalents of fullerene in step 1) (i.e., the molar amount of fullerene is 6 to 12 times that of hexabromophenylbenzene), maintaining the system for ten minutes, and then placing the system on a programmable temperature-controlled heating table and programming the temperature to uniformly decrease from 80 to 100° C. to 20 to 25° C. over 1 hour, precipitating dark brown crystals visible to the naked eye to obtain water-soluble fullerene;

[0035] Optionally, in step 2), the fullerene is 6 times the equivalent (i.e., the fullerene is 6 times the molar amount of hexabromophenylbenzene). The excess fullerene can allow each molecular cage to contain one fullerene, and the excess fullerene can be removed by filtration because it is insoluble in water.

[0036] In a seventh aspect, use of the water-soluble fullerene described in the fifth aspect or the water-soluble fullerene prepared by the preparation method described in the sixth aspect in the preparation of a drug or health product for treating oxidative stress diseases;

[0037] Optionally, the oxidative stress disease includes one or more of hypertension, aortic aneurysm, hypercholesterolemia, atherosclerosis, cardiac ischemia-reperfusion injury, myocardial infarction, heart failure and arrhythmia.

[0038] Beneficial effects

[0039] The present invention utilizes supramolecular non-covalent hydrophobic interactions to combine six molecules together to form a water-soluble cubic supramolecular cage. The resulting cage can be used to combine a C 60 The molecules are perfectly wrapped in the molecular cage, thereby increasing C 60 The water solubility of C 60 Provides new ideas and further verifies C 60 It plays an important role in preventing oxidative stress-related cardiovascular disease damage.

[0040] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and to implement it according to the contents of the specification, and to make the above and other purposes, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0042] Figure 1 The figure is a flow chart of the preparation of Example 1 of the present invention, wherein a is a schematic diagram of the reaction route; b is a 3D simulation flow chart.

[0043] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of compound M1 in Example 1 of the present invention;

[0044] Figure 3 The optical spectrum of the compound of Example 1 of the present invention, wherein a is 6M1 and the complex 6M1@C 60 At 25°C (1.6×10 -5 mol / L) in water, and C 60 At 25°C (2.6×10 -6 mol / L) in dichlorobenzene; b is the UV-visible spectrum of 6M1 and 6M1@C 60 (1.6×10 -5 mol / L) in water at 25℃.

[0045] Figure 4 Figure 1 is a diagram of the supramolecular cage structure of Example 1 of the present invention; wherein (a) is a 6M1 stick simulation diagram; (b) is a 6M1@C 60 Space-filling diagram; (for clarity, hydrogen atoms, H2O molecules and iodine ions are omitted); (c) is the Luban lock diagram; (d) is 6M1@C 60 Optimized structural simulation diagram of the complex; (e) Optical microscope image of 6M1 single crystal; (f) 6M1@C 60 Single crystal optical microscope image.

[0046] Figure 5The effects of 6M1 (20, 40, 60, 80 and 100 nM) on the viability of FMC84 cells were detected by CCK-8 colorimetry. The effects of 6M1@C60 (20, 40, 60, 80, 100 nm) on the viability of FMC84 cells were detected by CCK-8 colorimetry. The effects of H2O2 (200, 400, 600, 800 and 1000 μM) on the viability of FMC84 cells were detected by CCK-8 colorimetry. 60 Effects of hydrogen peroxide on FMC84 cell viability.

[0047] Figure 6 Figure 3 shows the detection of reactive oxygen species and cellular inflammation and apoptosis in Example 3 of the present invention. (a) The fluorescent probe DCFH-DA was used to detect intracellular ROS levels. Bar = 300 μm. (b) RT-qPCR was used to detect the expression of intracellular IL-6, tumor necrosis factor-α (TNF-α), and caspase-3. (c) Western blotting was used to detect the expression of intracellular IL-6, tumor necrosis factor-α (TNF-α), and caspase-3.

[0048] Figure 7 This is the cell apoptosis assay for Example 4 of the present invention; (a) Apoptotic cells were detected by Hoechst staining, bar = 150 μm. (b) Apoptosis was assessed by flow cytometry. Cells were stained with FITC-labeled Annexin V and the fluorescent dye PI. These data are expressed as the percentage of apoptotic cells relative to total cells.

[0049] Figure 8 The cardiac function of mice was detected by ultrasound in Example 5 of the present invention. The figure represents the cardiac function of mice in different groups, where sham is the sham operation group, IR is the ischemia-reperfusion model, and IR+L is the I / R model. The model group was injected with a low dose of drug (injection of 0.2mL 600nM 6M1@C 60 ); IR+H is the I / R model group injected with high dose of drug (injection of 0.2mL6000nM 6M1@C 60 ).

[0050] Figure 9 The Western blotting results of Example 6 of the present invention are shown in FIG. 6M1@C. 60Effects of 6M1@C60 on intracellular Akt, p-Akt, Nrf2, and HO-1 protein expression levels. (b) Effects of 6M1@C60 on cellular Akt, p-AKT, Nrf2, and HO-1 protein expression levels were observed after Akt inhibition with an Akt blocker (MK2206). (c) Immunofluorescence analysis of Nrf2 localization. NRF2 (red) labeled FMC84 cells, and DAPI (blue) stained cell nuclei. Bar = 75 μm (Data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, *P < 0.001, NS indicates not significant).

[0051] Figure 10 The results of intracellular ROS level detection after Nrf2 blocking in Example 7 of the present invention are shown in Figure 7. (a) The fluorescent probe DCFH-DA is used to detect intracellular ROS levels. (b) RT-qPCR is used to detect the expression of intracellular IL-6, tumor necrosis factor-α (TNF-α), and caspase-3. (c) Western blotting is used to detect the expression of intracellular IL-6, tumor necrosis factor-α (TNF-α), and caspase-3. DETAILED DESCRIPTION

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0053] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In some embodiments, raw materials, schemes, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0054] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0055] In the following examples, all raw materials used are commercially available, among which FMC84 cells were purchased from Jinan Yifei Biotechnology Co., Ltd., and hexabromophenylbenzene, p-vinylpyridine and PdCl2(PPh3)2 were purchased from Shanghai Titan Technology Co., Ltd.

[0056] The present invention adopts the classic Heck reaction to prepare a hexagonal amphiphilic molecule in one step, and then methylates it to prepare a high-yield amphiphile M1. The hydrophilic ends of M1 tend to stay together to form a hydrophilic outer periphery of the cube. The iodide ion, as the counteranion, remains on the outer surface of the cube and utilizes its non-covalent hydrophobic interaction in water to assemble and construct a supramolecular cage 6M1 with a hydrophobic cavity of about 1.4 nm in diameter. Since the cage cavity size is similar to C 60 Matching, the system was placed on a heating table with programmable temperature control, first preheated the saturated aqueous solution of M1 to 80℃~100℃, then added six times the equivalent of C 60 Solid, maintain the system for ten minutes, then cool it down to 20℃-25℃ evenly within 1 hour, and finally achieve the C 60 The package obtained was a water-soluble 60 Supramolecular cage 6M1@C 60 , thereby increasing C 60 water solubility.

[0057] Example 1

[0058] Water-soluble supramolecular cage 6M1@C 60 The preparation process is as follows Figure 1 shown

[0059] a) Under an inert gas atmosphere, 0.2 mmol of hexabromophenylbenzene, 2.4 mmol of p-vinylpyridine, 2.4 mmol of potassium carbonate, and 0.04 mmol of PdCl2(PPh3)2 were reacted in 10 mL of anhydrous N,N-dimethylformamide at 150°C for 48 hours, and the product 2, i.e., 6-vinylpyridylhexaphenylbenzene, was obtained after post-treatment;

[0060] b) Under inert gas conditions, 0.8 mmol of product 2 was dissolved in 10 mL of chloroform, 40 mmol of iodomethane was added, and the mixture was reacted at 45°C for 24 hours. After post-treatment, the final product M1 was obtained;

[0061] c) Heat the saturated aqueous solution of M1 at 80℃~100℃ to ensure that M1 is completely dissolved, and add six times the equivalent of C 60 Solid (ie C 60 The solid is 6 times the molar amount of hexabromophenylbenzene), the system is maintained for ten minutes, the system is placed on a heating table with programmable temperature control, and the program is set to uniformly reduce the temperature from 80℃ to 100℃ to 20℃-25℃ within 1 hour, dark brown crystals visible to the naked eye will precipitate, and the encapsulated C is obtained through the precise self-assembly of the amphiphile in the aqueous solution. 60 Water-soluble supramolecular cage 6M1@C 60 (Due to the successful package 6M1@C 60It is water soluble, and the rest is insoluble C 60 , then filtered to remove), supramolecular cage 6M1@C 60 The water solubility of is about 12 mg / ml;

[0062] The H NMR spectrum of compound M1 is as follows Figure 2 shown.

[0063] Figure 1 H NMR spectrum of compound M1 ( 1 H NMR) 1 H NMR (400MHz, DMSO-d6) δ8.78(d,J=6.9Hz,12H),8.05(d,J=6.4Hz,13H),7.72(d,J=1 6.3Hz, 6H), 7.33 (d, J = 7.9Hz, 18H), 7.09 (d, J = 7.9Hz, 12H), 4.21 (d, J = 8.1Hz, 19H).

[0064] Compound 6M1, C 60 , complex 6M1@C 60 The UV spectrum and fluorescence spectrum of Figure 3 As shown, the UV spectrum ( Figure 3 a) The results show that 6M1 and 6M1@C 60 There are obvious differences in the absorption peaks of 6M1@C after wrapping fullerene. 60 A broad peak appears in the curve of C 60 The presence of; At the same time, in the fluorescence spectrum ( Figure 3 b) 6M1@C 60 The fluorescence decrease is also observed in the curve of C 60 The molecules are enclosed in the 6M1 molecular cage. The above results show that 6M1 and C 60 Self-assembly in water was successful.

[0065] Figure 4 c is the Luban lock diagram, Figure 4 a is a stick figure simulation of 6M1, Figure 4 b is the space-filling simulation diagram of 6M1, Figure 4 d is 6M1@C 60 Optimized structural simulation diagram of the complex. Figure 4 e is an optical microscope image of a 6M1 single crystal. Compound 6M1 is a pale yellow hexahedral crystal. Figure 4 f is 6M1@C 60 Single crystal optical microscope image of the composite 6M1@C 60 It is a dark brown hexahedral crystal. Figure 4 The pale yellow hexahedral crystals in e are obviously different, indicating that the 6M1 cage encloses C60 .

[0066] Example 2

[0067] Cell proliferation was detected by CCK-8 assay. Mouse cardiomyocytes FMC84 were seeded in 96-well culture plates at a concentration of 3000 cells / well.

[0068] The treatment methods for each group are:

[0069] (a) Treatment group: 0, 20, 40, 60, 80, and 100 nM 6M1 were added to a 96-well culture plate and incubated for 6 h. Cell proliferation activity was detected by CCK-8 assay.

[0070] (b) Treatment group: 0, 20, 40, 60, 80, and 100 nm of 6M1@C were added to a 96-well culture plate. 60 After incubation for 6 h, the cell proliferation activity was detected by CCK-8 assay;

[0071] (c) Treatment group: 0, 200, 400, 600, 800, and 1000 μM H2O2 were added to a 96-well culture plate and incubated for 6 h, and the cell proliferation activity was detected by CCK-8 assay;

[0072] (d) Treatment group: 0, 20, 40, 60, 80, and 100 nm of 6M1@C were added to a 96-well culture plate. 60 , and 600 μM H2O2 was added to each well. A blank control group and a positive control group (only H2O2 was added) were set up. The cells were incubated for 6 h. The CCK-8 method was used to detect the expression of 6M1@C 60 Effects of hydrogen peroxide on FMC84 cell viability.

[0073] After each treatment, CCK-8 (10 μL) was added and incubated for another 1 hour. The absorbance at 450 nm was then measured.

[0074] Figure 5 The results show that: Figure 5 The results of a and 5b show that when 6M1 or 6M1@C 60 During incubation, cell survival was not affected, indicating that 6M1 or 6M1@C 60 The toxicity is low. Figure 5 c The results showed that the effect of H2O2 on cell viability was concentration-dependent. After 600μMH2O2 stimulated the cells for 6h, the subsequent detection resulted in a 40% to 50% decrease in cell viability compared with the control group. At the optimal hydrogen peroxide concentration and time, the addition of different concentrations of 6M1@C 60 . Figure 5 d The results showed that 60nM 6M1@C 60(Concentration within the system) can significantly improve H2O2-induced myocardial damage.

[0075] Among them, C 60 It is insoluble in water and was not used as a control.

[0076] In the following treatments, unless otherwise specified, the concentration of the drug added refers to the final concentration. For example, in Example 3, H2O2+6M1@C 60 600μM H2O2 and 60nM 6M1@C were added to the group 60 This means that in the 6-well plate of this group, the final concentration of H2O2 is 600μM, 6M1@C 60 The final concentration is 60 nM.

[0077] Example 3

[0078] Mouse cardiomyocytes (FMC84) were seeded into 6-well plates. When the density reached about 80%, the cells were divided into three groups: blank control group (no treatment), 6M1@C 60 Group (addition of 60nM 6M1@C alone) 60 ), H2O2 group (single addition of 600μM H2O2), H2O2+6M1@C 60 Group (addition of 600μM H2O2 and 60nM 6M1@C 60 ) for 6 hours. ROS levels were then detected using the fluorescent probe DCFH-DA, and the expression of intracellular IL-6, tumor necrosis factor-α, and caspase-3 was measured by RT-qPCR (quantitative analysis kit (AG11718) from Acry Biotech). Western blotting was used to detect the expression of intracellular IL-6, tumor necrosis factor-α, and caspase-3 (reagents purchased from Beyotime Biotech).

[0079] ROS levels were measured using the fluorescent probe DCFH-DA (reactive oxygen species detection kit (S0033S) purchased from Beyotime) diluted 1:1000 in serum-free culture medium to a final concentration of 10 μM. The original cell culture medium was removed and the diluted DCFH-DA was added. The cells were incubated at 37°C in a cell culture incubator for 30 minutes, washed three times with serum-free culture medium, and photographed under an inverted fluorescence microscope.

[0080] Figure 6 The results showed that H2O2 could significantly increase the level of ROS in cells. Figure 6 a indicates 6M1@C 60 The introduction of α-glucose significantly reduced the intracellular ROS level.

[0081] In addition, the effects of H2O2 on proinflammatory cytokines IL-6 and tumor necrosis factor-α were detected by RT-PCR and WB. Figure 6 b and 6c showed that H2O2 could increase the expression of IL-6 and tumor necrosis factor-α, while 6M1@C 60 This change can be blocked. Caspase3 is the most critical apoptosis execution protein in the process of cell apoptosis. WB detection data ( Figure 6 c) shows that H2O2 increased the expression of caspase3, while 6M1@C 60 Treatment inhibited the expression of caspase3 ( Figure 6 c).

[0082] Example 4

[0083] Mouse cardiomyocytes (FMC84) were seeded in 6-well plates. After the density reached about 80%, they were divided into groups for treatment: blank control group (no treatment), 6M1@C 60 Group (addition of 60nM 6M1@C alone) 60 ), H2O2 group (single addition of 600μM H2O2), H2O2+6M1@C 60 Group (addition of 600μM H2O2 and 60nM 6M1@C 60 ) for 6 hours. Apoptotic cells were then stained using the Beyotime Hoechst staining kit (C0003). The assay method was as follows: 0.5 ml of fixative was added to each well and fixed for 30 minutes. The fixative was discarded, and the cells were washed twice with PBS for 3 minutes each. 0.5 ml of Hoechst 33258 staining solution was then added for 5 minutes. After washing with PBS, the samples were observed and photographed.

[0084] Figure 7 The results showed that the Hoechst cell apoptosis detection kit and flow cytometry also showed that 6M1@C 60 Improved H2O2-induced cell apoptosis ( Figure 7 a and 7b). The above results show that 6M1@C 60 It has a protective effect on H2O2-induced oxidative stress damage.

[0085] Example 5

[0086] I / R model preparation: Male C57 mice (8 weeks old, purchased from Guangzhou Jinwei Biotechnology Co., Ltd., average weight approximately 25 g, total blood volume approximately 2 mL) were anesthetized with 2% isoflurane. The descending anterior coronary artery was ligated with 7-0 suture. A soft tube was placed under the ligature and tied with a slipknot to create an I / R model (ischemia-reperfusion model). The sham group did not undergo ligation; all other procedures were the same, and the chest cavity was sealed postoperatively.

[0087] Grouping and treatment method: The treatment group was injected with drugs through tail vein 30 minutes in advance (the high dose group (H) was injected with 0.2mL 6000nM 6M1@C 60 (Drug concentration in mouse blood is about 600nM), low-dose group (L) injected with 0.2mL 600nM 6M1@C 60 The drug concentration in mouse blood was approximately 60 nM. An equal volume of normal saline was injected into the tail vein of the model and sham groups 30 minutes before surgery. Left ventricular end-diastolic and end-systolic diameters (LVIDd and LVIDs), as well as ejection fraction and fractional shortening (LVEF and LVFS), were measured using the Teicholz method.

[0088] Figure 8 The results showed that compared with the sham group, the LVIDd and LVIDs values ​​of the model group (I / R) were significantly increased, and the LVEF and LVFS values ​​were significantly decreased (P<0.05). 60 The high-dose group (H) improved the cardiac function of mice more effectively than the low-dose group (L).

[0089] Example 6

[0090] Western blotting assay (WB) was used to detect protein levels of P-AKT, AKT, Nrf2, HO-1, and GAPDH (reagents purchased from Beyotime Biotechnology): After culture, cells were disrupted and total protein was extracted. Cellular protein content was determined using a BCA protein assay kit with RIPA buffer. The remaining extracted protein was added to loading buffer and boiled at 99°C for 10 min. The boiled protein was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated with antibodies against Nrf2 (1:800), HO-1 (1:1000), IL-6 (1:800), tumor necrosis factor-α (1:1000), caspase-3 (1:1000), and GAPDH (1:3000). Finally, horseradish peroxidase-conjugated secondary antibodies were used for incubation, and protein signals were detected using a chemiluminescent HRP substrate kit.

[0091] Nrf2 expression and nuclear translocation were detected using immunofluorescence. After culture, cells were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.2% Triton 100 for 5 minutes, and blocked with 1% BSA for 1 hour at room temperature. After washing with PBS, cells were incubated with a 1:200 dilution of Nrf2 primary antibody and 1:100 dilutions of IgGH and LAlexa Fluor 488 as secondary antibodies. Finally, nuclei were stained with DAPI to visualize staining.

[0092] Example 6.1

[0093] Mouse cardiomyocytes (FMC84) were seeded in 6-well plates and divided into three groups: blank control group (no treatment), 6M1@C 60 Group (addition of 60nM 6M1@C alone) 60 ), H2O2 group (single addition of 600μM H2O2), H2O2+6M1@C 60 Group (addition of 600μM H2O2 and 60nM 6M1@C 60 ), after 6 hours of treatment, the cells were broken and the total protein was extracted. The expression of P-AKT, AKT, Nrf2, HO-1 and GAPDH in the cells was detected by WB method (as shown in Figure 2). Figure 9 a), and the expression and nuclear translocation of Nrf2 in cells were detected by cell immunofluorescence (as shown in Figure 9 c).

[0094] Figure 9 aThe results showed that the expression levels of p-Akt, Nrf2 and HO-1 in the H2O2 group were increased, and the H2O2+6M1@C 60 6M1@C in the group 60 The participation of 6M1@C further promoted the increase of p-Akt, Nrf2 and HO-1 expression levels, suggesting that 60 May be involved in the regulation of Akt / Nrf2 / HO-1 signaling pathway.

[0095] Figure 9 cThe results show that 6M1@C 60 It can further promote the expression and nuclear translocation of Nrf2 under H2O2-induced oxidative stress.

[0096] Example 6.2 Effect of Akt pathway and protein expression:

[0097] To verify whether there is a regulatory relationship between the Akt pathway and the Nrf2 / HO-1 pathway, an Akt inhibitor (MK2206, purchased from Beyotime Reagent Co., Ltd., is a highly selective Akt 1 / 2 / 3 inhibitor. Pretreatment with MK2206 can significantly inhibit Akt phosphorylation) was used. The treatment method was based on the instructions for use of the MK2206 reagent on the Beyotime official website. The specific treatment was as follows:

[0098] Mouse cardiomyocytes (FMC84) were seeded in 6-well plates and divided into the following treatment groups: blank control group (no treatment), MK2206 group (pretreated with MK2206 for 12 hours), H2O2 group (not pretreated with MK2206, but only with 600 μM H2O2), H2O2+6M1@C 60 Group (no MK2206 pretreatment, 600 μM H2O2 and 60 nM 6M1@C 60 ),MK2206+H2O2+6M1@C 60 Group (pretreated with MK2206 for 12 hours and then treated with 600 μM H2O2 and 60 nM 6M1@C 60 ), after 6 hours of culture, the cells were broken and total protein was extracted, and the expression of P-AKT, AKT, Nrf2, HO-1 and GAPDH in the cells was detected by WB method as above. Figure 9 b.

[0099] Figure 9 b shows that after MK2206 was used to inhibit the phosphorylation of Akt, the expression of Nrf2 and HO-1 proteins also decreased in turn, indicating that in this example, the Nrf2 / HO-1 signaling pathway was activated by the Akt pathway, and the two were positively correlated.

[0100] Figure 9 b The results also showed that in the presence of H2O2, without the addition of MK2206 to inhibit Akt phosphorylation, 6M1@C 60 The expression of P-AKT, Nrf2 and HO-1 was further increased. After adding Akt inhibitor MK2206, 6M1@C 60 It cannot increase the expression of P-AKT, Nrf2, and HO-1, indicating that 6M1@C 60 It works by regulating the Nrf2 signaling pathway through the Akt signaling pathway.

[0101] Example 7

[0102] Mouse cardiomyocytes (FMC84) were seeded into 6-well plates. When the density reached about 80%, different treatments were performed on each group, including the following groups: blank control group (no treatment), H2O2 group (addition of 600 μM H2O2 without the inhibitor ML385), H2O2+ML385 group (pretreatment with the inhibitor ML385 and then addition of 600 μM H2O2), 6M1@C 60 +ML385 group (pre-treated with inhibitor ML385, then added with 60nM 6M1@C 60 )、H2O2+6M1@C 60 +ML385 group (pre-treated with inhibitor ML385, followed by addition of 600μM H2O2 and 60nM 6M1@C 60 ), 6 hours later, ROS level detection was performed (see Example 3 for detection method), and the results were as follows Figure 10 .

[0103] In the above treatment groups, the pretreatment time of the inhibitor ML385 was 6 hours. The pretreatment method of the inhibitor ML385 can be found in the instructions for use of the ML385 reagent of Selleck Company, which is used to block Nrf2.

[0104] Figure 10 The results showed that 6M1@C 60 It can reduce the levels of inflammatory indicators interleukin-6 and tumor necrosis factor-α and apoptosis indicator Caspase 3, and has a cell protective effect. After adding Nrf2 inhibitor ML385, 6M1@C 60 It cannot reduce the levels of inflammatory markers interleukin-6 and tumor necrosis factor-α and apoptosis marker Caspase 3, and does not play a cytoprotective role. 60 Nrf2 and HO-1 play a key role in the regulation of cell protection.

[0105] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and it is apparent that many changes and variations are possible in light of the foregoing teachings. The exemplary embodiments are selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and variations. Any simple modifications, equivalent variations, and modifications made to the exemplary embodiments described above are intended to fall within the scope of protection of the present invention.

Claims

1. A compound for hydrophilizing fullerene, having the structural formula (M1) shown below. 。 2. An intermediate of the compound according to claim 1, which is 6-vinylpyridylhexaphenylbenzene, having the structural formula shown in (2), 。 3. A method for preparing the intermediate according to claim 2, characterized in that: The steps include: Under an inert gas atmosphere, hexabromophenylbenzene and p-vinylpyridine react in a solvent in the presence of a weak base salt and a catalyst at 120°C-180°C to obtain a product of formula (2).

4. The preparation method according to claim 3, characterized in that The weak base salt is one or more of sodium carbonate, potassium carbonate and cesium carbonate.

5. The preparation method according to claim 3, characterized in that The catalyst is PdCl2(PPh3)2.

6. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of hexabromophenylbenzene to p-vinylpyridine is 1:(6~24).

7. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of hexabromophenylbenzene to p-vinylpyridine is 1:(8~16).

8. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of hexabromophenylbenzene to p-vinylpyridine is 1:(10~14).

9. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of hexabromophenylbenzene to p-vinylpyridine is 1:

12.

10. The preparation method according to any one of claims 3 to 5, characterized in that: The molar ratio of vinyl pyridine to weak base salt is 1: (0.8~1.4).

11. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of vinyl pyridine to weak base salt is 1: (1.0~1.2).

12. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of vinyl pyridine to weak base salt is 1:

1.

13. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of catalyst to hexabromophenylbenzene is (0.1-0.24):

1.

14. The preparation method according to any one of claims 3 to 5, characterized in that The molar ratio of the catalyst to the hexabromophenylbenzene is 0.2:

1.

15. The preparation method according to any one of claims 3 to 5, characterized in that The solvent is a polar solvent.

16. The preparation method according to any one of claims 3 to 5, characterized in that: The solvent was anhydrous N,N-dimethylformamide.

17. The preparation method according to any one of claims 3 to 5, characterized in that The reaction temperature is 130~180℃.

18. The preparation method according to any one of claims 3 to 5, characterized in that: The reaction temperature is 140~160℃.

19. The preparation method according to any one of claims 3 to 5, characterized in that: The reaction temperature was 150°C.

20. The preparation method according to any one of claims 3 to 5, characterized in that The reaction time is 40~60h.

21. The preparation method according to any one of claims 3 to 5, characterized in that The reaction time is 48 h.

22. A method for preparing the compound according to claim 1, characterized in that: Under inert gas conditions, in a solvent, the intermediate of formula (2) according to claim 2 or the intermediate of formula (2) prepared by the preparation method according to any one of claims 3 to 21 is reacted with iodomethane to produce a product of formula (M1).

23. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:(6-60).

24. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:(12~54).

25. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:(30-54).

26. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:(40-54).

27. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:(45-54).

28. The preparation method according to claim 22, characterized in that The molar ratio of the intermediate of formula (2) to methyl iodide is 1:

50.

29. The preparation method according to any one of claims 22 to 28, characterized in that The reaction temperature of the intermediate of formula (2) and methyl iodide is 35-60°C.

30. The preparation method according to any one of claims 22 to 28, characterized in that The reaction temperature of the intermediate of formula (2) and methyl iodide is 40-60°C.

31. The preparation method according to any one of claims 22 to 28, characterized in that The reaction temperature of the intermediate of formula (2) and methyl iodide is 40-50°C.

32. The preparation method according to any one of claims 22 to 28, characterized in that The reaction temperature of the intermediate of formula (2) and methyl iodide is 45°C.

33. The preparation method according to any one of claims 22 to 28, characterized in that The reaction time of the intermediate of formula (2) and methyl iodide is 20 to 36 hours.

34. The preparation method according to any one of claims 22 to 28, characterized in that The reaction time of the intermediate of formula (2) and methyl iodide is 24 hours.

35. The preparation method according to any one of claims 22 to 28, characterized in that In the reaction of the intermediate of formula (2) with iodomethane, the solvent is methyl halide.

36. The preparation method according to claim 35, characterized in that The solvent is chloroform.

37. A water-soluble fullerene, comprising a fullerene and a polyhedral molecular cage encapsulating the fullerene, wherein the polyhedral molecular cage is self-assembled by the compound M1 according to claim 1 or the compound M1 prepared by the preparation method according to any one of claims 22 to 36.

38. The water-soluble fullerene according to claim 37, characterized in that The polyhedral molecular cage is self-assembled by six compounds M1.

39. The water-soluble fullerene according to claim 37, wherein The solubility of water-soluble fullerene is 12 mg / ml.

40. The water-soluble fullerene according to claim 37, wherein The fullerene is C m , m is an integer between 30 and 90.

41. The water-soluble fullerene according to claim 37, wherein The fullerene is C 60 、C 70 One or more of the .

42. The water-soluble fullerene according to claim 37, wherein The polyhedral molecular cage has a hydrophobic cavity with a diameter of 1 to 2 nm.

43. A method for preparing water-soluble fullerene, characterized in that: The steps include: 1) heating a saturated aqueous solution of the compound M1 according to claim 1 or the compound M1 prepared by the preparation method of any one of claims 22 to 36 at 80° C. to 100° C.; 2) Add 6-12 times the equivalent of fullerene in step 1), maintain the system for ten minutes, and then uniformly cool the system from 80-100°C to 20-25°C over 1 hour to precipitate dark brown crystals visible to the naked eye, thereby obtaining water-soluble fullerene.

44. The preparation method according to claim 43, characterized in that In step 2), a heating stage with programmable temperature control is used for cooling.

45. The preparation method according to claim 43, characterized in that In step 2), the fullerene is 6 times equivalent.

46. ​​Use of the water-soluble fullerene according to any one of claims 37 to 42 or the water-soluble fullerene prepared by the preparation method according to any one of claims 43 to 45 in preparing a drug for treating oxidative stress diseases.

47. The use according to claim 46, characterized in that The oxidative stress diseases include one or more of hypertension, aortic aneurysm, hypercholesterolemia, atherosclerosis, cardiac ischemia-reperfusion injury, myocardial infarction, heart failure and arrhythmia.

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