GLUT inhibitor based on disulfide death mechanism and preparation method thereof

Abstract

The invention provides a GLUT inhibitor based on a disulfide death mechanism, the molecular formula of the GLUT inhibitor is C17H18N4O2S2, the molecular weight is 378.48, the GLUT inhibitor comprises a core structure containing a sulfur group, and the core structure and specific amino acid residues in GLUT protein form covalent bonds; the preparation method of the GLUT inhibitor comprises the following steps: S1, mixing sodium diethyldithiocarbamate and copper chloride in absolute ethyl alcohol, and reacting at the temperature of 25 DEG C for 24 hours to obtain a disulfiram copper compound; and S2, mixing the disulfiram copper compound and glucose transporter in a phosphate buffer solution, and reacting at 37 DEG C for 12 hours to obtain the GLUT inhibitor based on the disulfide death mechanism. The core structure of the inhibitor can form a covalent bond with a specific amino acid residue in a GLUT protein, so that the activity of the GLUT is irreversibly inhibited, the activity of the GLUT is easily approached to the GLUT protein, and due to the structural characteristics, the inhibitor can efficiently and specifically recognize and inhibit the GLUT protein and induce tumor cells to generate dithio death.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a GLUT inhibitor based on a disulfide death mechanism and a preparation method thereof. BACKGROUND

[0002] Tumor cells are highly dependent on glucose for energy supply, and the GLUT inhibitor can specifically inhibit the growth and proliferation of tumor cells, and induce disulfide death, thereby providing a new strategy and method for tumor treatment.

[0003] Current tumor treatment methods mainly include surgery, chemotherapy, radiotherapy and targeted therapy. Surgical resection of tumor tissue is suitable for early-stage tumors, but it is difficult to remove micro metastases. Chemotherapy uses chemical drugs to kill cancer cells, but there is a problem of drug resistance. Cancer cells can make drugs ineffective through various mechanisms, such as overexpression of drug efflux pumps and mutation of drug action targets. Radiotherapy can cause damage to surrounding normal tissues, and some tumors are not sensitive to radiotherapy. These problems make tumor treatment face many challenges, and new treatment strategies and methods are urgently needed.

[0004] In view of the above-mentioned deficiencies in actual production and implementation, the present application is modified and improved, and in the spirit of seeking good and the idea of seeking good, and with the aid of professional knowledge and experience, and after many trials and experiments, the present application is created, and a GLUT inhibitor based on a disulfide death mechanism and a preparation method thereof are provided. SUMMARY

[0005] The present application provides a GLUT inhibitor based on a disulfide death mechanism and a preparation method thereof, which solves the problems in the prior art.

[0006] The technical scheme of the present application is as follows: a GLUT inhibitor based on a disulfide death mechanism, the molecular formula of which is C 17 H 18 N4O2S2, the molecular weight is 378.48, and it contains a core structure containing a sulfur-containing group, which forms a covalent bond with a specific amino acid residue in the GLUT protein.

[0007] A preparation method of a GLUT inhibitor, comprising the following steps:

[0008] S1: mixing sodium diethyldithiocarbamate and copper chloride with copper chloride in anhydrous ethanol, the reaction temperature is 25℃, and the reaction time is 24 hours, to obtain a disulfiram copper complex;

[0009] S2: mixing the disulfiram copper complex with a glucose transporter in a phosphate buffer, the reaction temperature is 37℃, and the reaction time is 12 hours, to obtain a GLUT inhibitor based on a disulfide death mechanism.

[0010] As a preferred embodiment, 5.0 g of the sodium diethyldithiocarbamate is selected and placed in a 250 mL beaker, and dissolved in 100 mL of anhydrous ethanol.

[0011] As a preferred embodiment, the disulfiram copper complex is filtered with a Buchner funnel, and the obtained solid is washed with 10 mL of anhydrous ethanol for 3 times.

[0012] As a preferred embodiment, 5.0 g of the disulfiram copper complex is selected and dissolved in 50 mL of a phosphate buffer, and 0.2 g of a glucose transporter is added, and the phosphate buffer has a pH of 7.4.

[0013] As a preferred embodiment, after the GLUT inhibitor based on the disulfiram death mechanism is precipitated for 2 hours, it is placed in a vacuum drying oven for drying, the temperature is 40 DEG C, and the drying time is 12 hours.

[0014] After the above technical solutions are adopted, the tumor cells often produce drug resistance through mechanisms such as overexpression of drug efflux pumps. The action target of the GLUT inhibitor is GLUT protein, which has a special structure and is different from the traditional drug action target, and is not easily affected by the drug efflux pump. The core structure of the inhibitor containing a sulfur group can form a covalent bond with specific amino acid residues of the GLUT protein, realize irreversible inhibition, and make it difficult for the tumor cells to change the GLUT structure to escape the action of the drug.

[0015] The inhibitor can specifically block the function of GLUT, so that the tumor cells cannot normally obtain glucose, and further interfere with their energy metabolism and redox balance. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, without creative labor, other drawings can also be obtained from these drawings.

[0017] Fig. 1 The schematic diagram of the influence of different treatments on protein expression under non-reducing and reducing conditions of the present application;

[0018] Fig. 2 The tumor volume-time curve of the present application. DETAILED DESCRIPTION

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1

[0021] like Figs. 1-2 As shown, a GLUT inhibitor based on the disulfide death mechanism has the molecular formula C 17 H 18 N4O2S2, with a molecular weight of 378.48, is an inhibitor primarily composed of a core structure containing a sulfur-containing group. This core structure can form covalent bonds with specific amino acid residues in the GLUT protein, thereby irreversibly inhibiting GLUT activity. In addition, the inhibitor also contains a hydrophilic group and a hydrophobic group. The hydrophilic group facilitates the interaction of the inhibitor with water molecules on the cell membrane, making it more accessible to the GLUT protein; while the hydrophobic group interacts with the lipid bilayer of the cell membrane, enhancing the inhibitor's stability within the cell membrane. This structural feature allows the inhibitor to efficiently and specifically recognize and inhibit the GLUT protein, inducing disulfide death in tumor cells.

[0022] A method for preparing a GLUT inhibitor includes the following steps:

[0023] To prepare the GLUT inhibitor based on the disulfide death mechanism, first accurately weigh 5.0 g of sodium diethyldithiocarbamate (NADDC) into a 250 mL beaker, add 100 mL of anhydrous ethanol to dissolve it. Then, slowly add 2.0 g of copper chloride (CuCl2) to it, and continuously stir to fully react the two. The reaction process is carried out in a constant temperature water bath at 25°C for 24 hours. After the reaction is completed, use a Buchner funnel to filter, and wash the obtained solid with anhydrous ethanol 3 times, 10 mL each time, to remove impurities. Dry the washed solid in a vacuum drying oven, set the temperature to 40°C, and dry for 12 hours to obtain the disulfiram copper complex (Cu[DDC]2). Next, dissolve 0.5 g of Cu[DDC]2 in 50 mL of phosphate buffer (pH = 7.4), and add 0.2 g of glucose transporter (GLUT), and react in a water bath at 37°C for 12 hours. After the reaction is completed, centrifuge at 8000 r / min for 10 minutes at 4°C to separate the precipitate. Wash the precipitate with distilled water 3 times, 10 mL each time, and finally dry the precipitate in a vacuum drying oven at 40°C for 12 hours to obtain the GLUT inhibitor based on the disulfide death mechanism. Control the reaction temperature and time during preparation to ensure the purity and activity of the inhibitor, and ensure that the prepared inhibitor has good biological activity and stability.

[0024] The experimental data are as follows:

[0025] Take tumor cells in the logarithmic growth phase, such as malignant glioblastoma cells, and inoculate them into a 96-well plate at a density of 1 x 105 cells per well, and culture for 24 hours. After the cells adhere, add different concentrations of the inhibitor (0 μM, 10 μM, 20 μM, 40 μM, 80 μM), with 3 replicate wells for each concentration, and set up a blank control group. Continue to culture for 48 hours, and then use the MTT method to detect cell survival rate. Add 20 μL of MTT solution (5 mg / mL) to each well, continue to culture for 4 hours, then carefully aspirate the culture medium in the well, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the crystals. Finally, use a microplate reader to measure the absorbance value of each well at 490 nm, and calculate the cell survival rate. The results show that as the concentration of the inhibitor increases, the survival rate of tumor cells significantly decreases, and when the concentration of the inhibitor is 80 μM, the cell survival rate is only about 20%. This proves that the inhibitor can effectively inhibit the growth of tumor cells and has good anti-tumor effect.

[0026] By specifically blocking the GLUT function, interfering with the energy metabolism and redox balance of tumor cells, and inducing disulfide death, the treatment effect of tumors can be improved, new treatment methods can be provided for tumor patients, the survival period of patients can be prolonged, and the quality of life can be improved. At the same time, it also opens up a new direction for the research and development of the field of tumor treatment.

[0027] The application provides a GLUT inhibitor based on a double sulfur death mechanism and a preparation method thereof, and research and development in the field of tumor treatment bring new ideas, by simultaneously inhibiting glucose uptake and inducing cell death, tumor cells can be more effectively targeted, and the toxic side effects on normal cells are reduced, the above only is the preferred embodiment of the application, it should be pointed out that, for ordinary skilled in the art, without departing from the method of the application, a number of improvements and supplements can also be made, these improvements and supplements should also be regarded as the protection scope of the application.

[0028] The application provides a GLUT inhibitor based on a double sulfur death mechanism and a preparation method thereof, the above is only the preferred embodiment of the application, and does not limit the application, any modification, equivalent replacement, improvement and the like made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A GLUT inhibitor based on the dual sulfide death mechanism, the molecular formula is C 17 H 18 N4O2S2, the molecular weight is 378.48, characterized in that, The core structure comprising a sulfur-containing group forms a covalent bond with specific amino acid residues in the GLUT protein.

2. The method of claim 1, wherein the GLUT inhibitor is prepared by, The method comprises the following steps: S1: mixing sodium diethyldithiocarbamate and copper chloride in anhydrous ethanol, the reaction temperature is 25°C, and the reaction time is 24 hours to obtain a disulfiram copper complex; S2: mixing the disulfiram copper complex and glucose transporter in a phosphate buffer, the reaction temperature is 37°C, and the reaction time is 12 hours to obtain a GLUT inhibitor based on a disulfide death mechanism.

3. The method for preparing a GLUT inhibitor according to claim 2, characterized in that, 5.0 g of the sodium diethyldithiocarbamate and copper chloride are selected and placed in a 250 mL beaker, and then mixed and dissolved in 100 mL of anhydrous ethanol.

4. The method for preparing a GLUT inhibitor according to claim 2, characterized in that, The disulfiram copper complex is filtered through a Buchner funnel, and the obtained solid is washed with anhydrous ethanol for 3 times, each time with 10 mL.

5. The method for preparing a GLUT inhibitor according to claim 2, characterized in that, 5.0 g of the disulfiram copper complex is selected and dissolved in 50 mL of a phosphate buffer, and then 0.2 g of glucose transporter is added, and the pH of the phosphate buffer is 7.

4.

6. The method for preparing a GLUT inhibitor according to claim 2, characterized in that, After the GLUT inhibitor based on the disulfide death mechanism is precipitated for 2 hours, it is placed in a vacuum drying oven and dried at a temperature of 40°C for 12 hours.

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