Anti-tumor composition containing N-acetyl-D-glucosamine and preparation method thereof

Abstract

The invention discloses an anti-tumor composition containing N-acetyl-D-glucosamine and a preparation method of the anti-tumor composition in the field of anti-tumor preparations. The anti-tumor composition comprises the following components in parts by weight: 1-3 parts of functionalized N-acetyl-D-glucosamine and 3-5 parts of a polypeptide modifier. Through polypeptide modification and application of the N-acetyl-D-glucosamine, the N-acetyl-D-glucosamine (GlcNAc) as a bioactive sugar molecule can possibly have the following effects in tumor microenvironment regulation and control through the immune regulation function of the N-acetyl-D-glucosamine; glycometabolism interference: cancer cells usually grow rapidly depending on the Warburg effect, and the functionalized N-acetyl-D-glucosamine can inhibit proliferation of the cancer cells by influencing the glycometabolism of the cancer cells, and can synergistically play an anti-tumor role in three directions of glycometabolism interference, immunoregulation and cell apoptosis induction.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor preparations, and specifically refers to an anti-tumor composition containing N-acetyl-D-glucosamine and a preparation method thereof. Background Art

[0002] N-acetyl-D-glucosamine (GlcNAc for short) is a derivative of glucose and widely exists in the components of the human cell wall, especially as a constituent of glycosaminoglycans and glycoproteins. It has been found that N-acetyl-D-glucosamine and its derivatives not only play an important role in immune regulation but may also have certain therapeutic effects against tumors. The inhibitory effect of N-acetyl-D-glucosamine on tumors is mainly achieved through the following mechanisms: Immunomodulatory effect: N-acetyl-D-glucosamine can enhance the anti-tumor immune response by strengthening the immune system function. For example, it can enhance the phagocytic function of macrophages, promote the activity of natural killer cells (NK cells), and regulate the function of T cells, enhancing the body's ability to recognize and eliminate tumor cells; Inhibition of tumor cell proliferation: Some studies have shown that N-acetyl-D-glucosamine can inhibit the proliferation of tumor cells by regulating the expression of cyclin. Specifically, it may reduce the division and proliferation of tumor cells by affecting certain signaling pathways (such as PI3K / Akt, MAPK, etc.); Induction of tumor cell apoptosis: N-acetyl-D-glucosamine can also inhibit tumor growth by inducing tumor cell apoptosis. Research has shown that it can activate the signaling pathways related to apoptosis and increase the incidence of tumor cell death; Inhibition of tumor angiogenesis: The growth and metastasis of tumors often rely on angiogenesis. N-acetyl-D-glucosamine has been shown in some studies to inhibit the formation of new blood vessels in tumors, thereby inhibiting the spread of tumors. In anti-tumor treatment, common side effects mainly come from chemotherapy, radiotherapy, and the use of other anti-tumor drugs. The following are some common side effects: Gastrointestinal side effects: Chemotherapy drugs often cause gastrointestinal symptoms such as nausea, vomiting, loss of appetite, and diarrhea. N-acetyl-D-glucosamine may help relieve these discomforts through its immunomodulatory effect, especially its protective effect on the gastrointestinal tract; Immune system suppression: Chemotherapy and some targeted therapy drugs can suppress the immune system and increase the risk of infection. N-acetyl-D-glucosamine helps enhance the function of the immune system and promote the activity of immune cells, thus helping to reduce the side effects of immune suppression; Myelosuppression: Many anti-tumor drugs can cause myelosuppression, which in turn leads to anemia, leukopenia, or thrombocytopenia. The combined use with N-acetyl-D-glucosamine may help relieve these adverse reactions because N-acetyl-D-glucosamine may promote cell repair and regeneration; Through reasonable combination use, N-acetyl-D-glucosamine is expected to reduce these common side effects in anti-tumor treatment, enhance the treatment effect, and improve the quality of life of patients. Summary of the Invention

[0003] In view of the above situation, to overcome the defects of the prior art, the present invention provides an anti-tumor composition containing N-acetyl-D-glucosamine and a preparation method thereof. Through polypeptide modification and the application of N-acetyl-D-glucosamine, the immunomodulatory function of N-acetyl-D-glucosamine can be utilized. As a bioactive sugar molecule, N-acetyl-D-glucosamine (GlcNAc) may play the following roles in the regulation of the tumor microenvironment: glycolytic interference. Cancer cells usually rely on glycolysis (Warburg effect) for rapid growth. Functionalized N-acetyl-D-glucosamine can inhibit the proliferation of cancer cells by affecting their glycolysis, and can synergistically exert anti-tumor effects in three directions: glycolytic interference + immunomodulation + apoptosis induction.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides an anti-tumor composition containing N-acetyl-D-glucosamine. The preparation raw materials of the composition include the following components in parts by weight: 1-3 parts of functionalized N-acetyl-D-glucosamine and 3-5 parts of polypeptide modifier;

[0005] Preferably, the preparation method of the functionalized N-acetyl-D-glucosamine specifically includes the following steps:

[0006] S1. Dissolve D-glucosamine in dichloromethane, place it in an environment of 0 °C, add triethylamine, stir and mix for 10 min, then slowly dropwise add a chloroformic acid-2,2,2-trichloroethyl ester / dichloromethane solution. While maintaining the condition of 0 °C, react for 30-50 min. After raising the reaction temperature to room temperature, continue to stir and react for 2-3 h. Adjust the reaction pH to neutral with saturated sodium bicarbonate solution, add dichloromethane for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain N-Troc glucosamine;

[0007] Preferably, in step S1, the weight concentration of D-glucosamine in dichloromethane is 40-60 mg / mL;

[0008] Preferably, in step S1, the mass-volume ratio of D-glucosamine to triethylamine is 0.7-0.9 g / mL;

[0009] Preferably, in step S1, the mass-volume ratio of D-glucosamine to chloroformic acid-2,2,2-trichloroethyl ester is 1-1.2 g / mL;

[0010] S2. Dissolve the N-Troc-glucosamine prepared in step S1 in dichloromethane. Under an ice-water bath condition, dropwise add hydrobromic acid - acetic acid solution, and stir at a speed of 120 - 180 rpm. After reacting for 2 - 4 h, add deionized water to quench the reaction. After neutralizing the reaction system to neutral with saturated sodium bicarbonate aqueous solution, add dichloromethane for extraction. Collect the organic phase, wash it with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify it to obtain bromo-substituted N-Troc-glucosamine;

[0011] Preferably, in step S2, the mass concentration of the N-Troc-glucosamine in dichloromethane is 20 - 40 mg / mL;

[0012] Preferably, in step S2, the volume ratio of hydrobromic acid to acetic acid in the hydrobromic acid - acetic acid solution is 1:2 - 3;

[0013] Preferably, in step S2, the mass-to-volume ratio of the N-Troc-glucosamine to the hydrobromic acid - acetic acid solution is 0.05 - 0.15 g / mL;

[0014] S3. Take the bromo-substituted N-Troc-glucosamine prepared in step S2 and dissolve it in DMF. Add sodium azide and mix evenly. Stir at a speed of 200 - 300 rpm at room temperature. After reacting for 30 - 60 min, add water to quench the reaction. After adding ethyl acetate, collect the organic phase, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain azido-α-D-glucose;

[0015] Preferably, in step S3, the mass concentration of the bromo-substituted N-Troc-glucosamine in DMF is 10 - 30 mg / mL;

[0016] Preferably, in step S3, the mass ratio of the bromo-substituted N-Troc-glucosamine to sodium iodide is 5:2 - 4;

[0017] S4. Dissolve the azido-α-D-glucose prepared in step S3 in anhydrous methanol, add glacial acetic acid, mix evenly, then add zinc powder, and react at 50 - 70 °C for 2 - 4 h. Filter, collect the filtrate, transfer it to an ice-water bath condition, add acetic anhydride, and stir and react at a speed of 150 - 180 rpm. After reacting for 2 - 3 h, add saturated sodium bicarbonate aqueous solution to adjust the pH to neutral, then add ethyl acetate for extraction, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain functionalized N-acetyl-D-glucosamine;

[0018] Preferably, in step S4, the mass concentration of α-D-glucose azide in anhydrous methanol is 0.05 - 0.1 g / mL;

[0019] Preferably, in step S4, the volume ratio of glacial acetic acid to anhydrous methanol is 0.75 - 1.2:1;

[0020] Preferably, in step S4, the added mass of zinc is 10 - 30% of the mass of α-D-glucose azide;

[0021] Preferably, in step S4, the mass-volume ratio of α-D-glucose azide to acetic anhydride is 10 - 50 mg / mL;

[0022] Preferably, the preparation method of the polypeptide modifier specifically includes the following steps:

[0023] A1. Immerse the Rink Amide Resin in DCM for swelling, remove the solvent, transfer the Rink Amide Resin to a pyridine / DMF solution, stir and react for 5 - 10 min, remove the pyridine / DMF solvent, add a new pyridine / DMF solvent and continue to stir and react for 10 - 15 min, then wash the Rink Amide Resin with DMF to obtain the deprotected Rink Amide Resin;

[0024] Preferably, in step A1, the volume fraction of pyridine in DMF in the pyridine / DMF solution is 15% - 25%;

[0025] A2. Dissolve Fmoc-Pbf-arginine in DMF, add HBTU and HOBt, mix well, add DIEA, continue to stir at a speed of 150 - 180 rpm for 3 - 5 min, then add the deprotected Rink Amide Resin prepared in step A1, and stir and react at a speed of 200 - 300 rpm at room temperature for 1 - 2 h, then filter, collect the Rink Amide Resin, and wash it repeatedly with DMF to obtain the Rink Amide Resin - oligomeric arginine;

[0026] Preferably, in step A2, the mass concentration of Fmoc-Pbf-arginine in DMF is 30 - 50 mg / mL;

[0027] Preferably, in step A2, the mass ratio of Fmoc-Pbf-arginine, HBTU and HOBt is 1.5 - 2:1:0.2 - 0.6;

[0028] Preferably, in step A2, the mass-volume ratio between the Fmoc-Pbf-arginine and DIEA is 2.0 - 2.5 g / mL;

[0029] A3. Dissolve p-carboxybenzenesulfonamide in DMF, add thionyl chloride dropwise under an ice-water bath, stir at a speed of 200 - 300 rpm overnight at room temperature, after detecting the reaction is complete by TLC, remove the solvent to obtain intermediate A. Dissolve intermediate A in DMF, add it to the reaction system in step A2, add TEA to keep the pH of the reaction system at 8 - 9, react at 25 - 40 °C for 4 - 8 h, then add ethyl acetate for extraction, collect the organic phase, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain Rink Amide Resin - oligoarginine - benzenesulfonamide;

[0030] Preferably, in step A3, the mass concentration of p-carboxybenzenesulfonamide in DMF is 40 - 50 mg / mL;

[0031] Preferably, in step A3, the mass-volume ratio between p-carboxybenzenesulfonamide and thionyl chloride is 0.2 - 0.25 g / mL;

[0032] A4. Place the Rink Amide Resin - oligoarginine - benzenesulfonamide prepared in step A3 in a TFA cleavage solution, stir and react at a speed of 150 - 180 rpm for 2 - 3 h, then filter, collect the filtrate, add pre-cooled anhydrous ether for precipitation, after centrifugation, discard the supernatant, collect the precipitate, dry it to obtain oligoarginine - intermediate A;

[0033] Preferably, in step A4, the volume of the TFA cleavage solution is 100 - 150 mL;

[0034] A5. Dissolve 4-pentynoic acid in dichloromethane, place it in an ice-water bath, add thionyl chloride, raise the temperature to room temperature, stir at a speed of 200 - 300 rpm for 12 - 16 h, then add potassium carbonate, transfer it to ice water, add it to the oligoarginine - benzenesulfonamide prepared in step A4, stir and react at a speed of 200 - 300 rpm at room temperature for 8 - 12 h, then add ethyl acetate for extraction, collect the organic phase, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain the polypeptide modifier;

[0035] Preferably, in step A5, the mass concentration of 4-pentynoic acid in dichloromethane is 40 - 50 mg / mL;

[0036] Preferably, in step A5, the mass-volume ratio between the 4-pentynoic acid and thionyl chloride is 0.1-0.15 g / mL:

[0037] Preferably, in step A5, the mass ratio between the 4-pentynoic acid and potassium carbonate is 1:2.5-4;

[0038] The present invention also provides a preparation method of an anti-tumor composition containing N-acetyl-D-glucosamine, specifically including the following steps:

[0039] ① Dissolve the functionalized N-acetyl-D-glucosamine and the polypeptide modifier in a tert-butanol / water solution according to parts by weight, add sodium ascorbate and copper sulfate pentahydrate, stir and react at 80-90 °C for 2-3 h, then add ethyl acetate for extraction. After collecting the organic phase, dry it with anhydrous sodium sulfate, and after purification, obtain the intermediate product B;

[0040] Preferably, in step ①, the mass concentration of the functionalized N-acetyl-D-glucosamine in the tert-butanol / water solution is 20-30 mg / mL;

[0041] Preferably, in step ①, in the tert-butanol / water solution, the mass ratio between tert-butanol and deionized water is 2-3:1;

[0042] Preferably, in step ①, the mass ratio among the functionalized N-acetyl-D-glucosamine, sodium ascorbate and copper sulfate pentahydrate is 10:1-3:2-3;

[0043] ② Dissolve the intermediate product B prepared in step ① in acetone, add a methanol solution of sodium methoxide, stir and react at room temperature for 30-50 min, then adjust the pH to neutral. Solids will precipitate out. Filter, collect the solids, and after purification, obtain the composition containing N-acetyl-D-glucosamine;

[0044] Preferably, in step ②, the volume of the acetone is 10-20 mL;

[0045] Preferably, in step ②, the addition amount of sodium methoxide is 10-16 mg.

[0046] The beneficial effects achieved by the present invention are as follows:

[0047] The present invention provides an anti-tumor composition containing N-acetyl-D-glucosamine and a preparation method thereof. Through polypeptide modification and the application of N-acetyl-D-glucosamine, the immunomodulatory function of N-acetyl-D-glucosamine can be utilized. As a bioactive sugar molecule, N-acetyl-D-glucosamine (GlcNAc) may play the following roles in the regulation of the tumor microenvironment: interference with glucose metabolism: cancer cells usually rely on glucose metabolism (Warburg effect) for rapid growth. Functionalized N-acetyl-D-glucosamine can inhibit the proliferation of cancer cells by affecting their glucose metabolism, and can synergistically exert anti-tumor effects in three directions: interference with glucose metabolism + immunomodulation + induction of apoptosis. N-acetyl-D-glucosamine affects the glucose metabolism and microenvironment of cancer cells, weakening the activity of cancer cells. The polypeptide modifier enhances the drug penetration ability and may promote the death of cancer cells by inhibiting tumor signaling pathways. The combination of the two may improve the anti-cancer effect through immune enhancement and tumor targeting; hindrance of glycosylation: the abnormal expression of glycosylated proteins on the surface of tumor cells can affect tumor metastasis and immune escape. N-acetyl-D-glucosamine can interfere with glycosylation modification and reduce the invasion ability of cancer cells. Among the polypeptide modifiers, oligol-arginine can enhance the cell membrane penetration ability (CPP, CellPenetratingPeptide), help the drug enter cancer cells, and improve the therapeutic effect. Oligol-arginine can enhance cellular oxidative stress and trigger the apoptosis pathway, causing cancer cells to self-destruct. The benzenesulfonamide moiety may reduce the survival signal of cancer cells by inhibiting protein tyrosine phosphatases (PTPs). Benzenesulfonamide modification may bind to tumor-related proteins and improve the drug targeting ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 1H NMR spectrum image of the compound in Example 1 of the present invention;

[0049] Figure 2 Inhibitory activity results graph of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention against carbonic anhydrase;

[0050] Figure 3 Inhibitory activity results graph of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention against HCT116.

[0051] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0053] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred methods and materials described herein are for illustrative purposes only and do not limit the content of this application.

[0054] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the test materials and test strains used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0055] Example 1

[0056] This example provides an anti-tumor composition containing N-acetyl-D-glucosamine, and the composition includes the following components in parts by weight: 1 part of functionalized N-acetyl-D-glucosamine and 3 parts of polypeptide modifier;

[0057] The preparation method of functionalized N-acetyl-D-glucosamine specifically includes the following steps:

[0058] S1. Take 1.2 g of D-glucosamine and dissolve it in 30 mL of dichloromethane. Place it in an environment of 0 °C, add 1.5 mL of triethylamine, stir and mix for 10 min, then take 0.8 mL and mix it evenly with 5 mL of dichloromethane to obtain a chloroformic acid 2,2,2-trichloroethyl ester / dichloromethane solution. Slowly drop the chloroformic acid 2,2,2-trichloroethyl ester / dichloromethane solution, keep the reaction at 0 °C, react for 30 - 50 min, raise the reaction temperature to room temperature, continue to stir and react for 2 - 3 h, adjust the reaction pH to neutral with saturated sodium bicarbonate solution, add dichloromethane for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain N-Troc glucosamine;

[0059] S2. Dissolve the N-Troc-glucosamine prepared in step S1 in 15 mL of dichloromethane. Under an ice-water bath condition, mix hydrobromic acid and acetic acid according to a volume ratio of 1:2.5, dropwise add the hydrobromic acid - acetic acid solution, stir at a speed of 150 rpm, after reacting for 3 h, add deionized water to quench the reaction. After neutralizing the reaction system to neutral with saturated sodium bicarbonate aqueous solution, add dichloromethane for extraction, collect the organic phase, wash it with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain bromo-substituted N-Troc-glucosamine;

[0060] S3. Take 0.5 g of the bromo-substituted N-Troc-glucosamine prepared in step S2 and dissolve it in 25 mL of DMF, add 0.3 g of sodium azide and mix evenly. Stir at a speed of 250 rpm under room temperature condition, after reacting for 45 min, add water to quench the reaction. After adding ethyl acetate, collect the organic phase, wash it with saturated sodium chloride solution, dry the separated organic phase with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify to obtain azido-α-D-glucose;

[0061] S4. Dissolve 1 g of the azido-α-D-glucose prepared in step S3 in 15 mL of anhydrous methanol, add 18 mL of glacial acetic acid, mix evenly, then add 0.2 g of zinc powder, place it at 60 °C and react for 3 h. Filter, collect the filtrate, then add 20 mL of acetic anhydride, stir and react at a speed of 180 rpm. After reacting for 2 h, add saturated sodium bicarbonate aqueous solution to adjust the pH to neutral, add ethyl acetate for extraction, wash it with saturated sodium chloride solution, dry the separated organic phase with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify to obtain functionalized N-acetyl-D-glucosamine;

[0062] A method for preparing a polypeptide modifier specifically includes the following steps:

[0063] A1. Immerse the Rink Amide Resin in DCM for swelling for 30 min, then remove the solvent. Transfer the Rink Amide Resin to a 15 vol% pyridine / DMF solvent, stir and react for 5 min, then remove the pyridine / DMF solvent, add a new pyridine / DMF solvent and continue to stir and react for 15 min, and then wash the Rink Amide Resin with DMF to obtain the deprotected Rink Amide Resin;

[0064] A2. Place 1 g of Fmoc-Pbf-arginine in a flask, add 20 mL of DMF to fully dissolve Fmoc-Pbf-arginine, add 0.5 g of HBTU and 0.2 g of HOBt, mix at a speed of 150 rpm. After mixing evenly, add 0.4 mL of DIEA, continue to mix for 5 min, then add the Rink Amide Resin prepared in step A1. Place it at room temperature and stir and react at a speed of 200 rpm. After reacting for 2 h, filter, collect the solid Rink Amide Resin, and wash the Rink Amide Resin repeatedly with DMF to obtain Rink Amide Resin-oligomeric arginine;

[0065] A3. Accurately weigh 0.8 g of p-carboxybenzenesulfonamide and place it in a flask, add 20 mL of DMF to fully dissolve p-carboxybenzenesulfonamide. Transfer the reaction system to an ice-water bath, add 3.2 mL of thionyl chloride, place it at room temperature, stir at a speed of 300 rpm. After stirring and reacting overnight, use TLC to detect that the reaction is complete, then distill off the excess DMF under reduced pressure to obtain intermediate A. Add 30 mL of DMF to redissolve intermediate A, mix it with the Rink Amide Resin-oligomeric arginine in step A2, add triethanolamine to adjust the pH of the reaction system to 8.5, and react at 25 °C for 8 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain Rink Amide Resin-oligomeric arginine-benzenesulfonamide;

[0066] A4. Place the Rink Amide Resin-oligomeric arginine-benzenesulfonamide prepared in step A3 in 150 mL of TFA cleavage solution, stir and react at a speed of 180 rpm for 2 h, then filter, collect the filtrate, add pre-cooled anhydrous ether for precipitation, centrifuge, discard the supernatant, collect the precipitate, and dry it to obtain oligomeric arginine-benzenesulfonamide;

[0067] A5. Accurately weigh 0.5 g of 4-pentynoic acid and dissolve it in 10 mL of dichloromethane. Transfer it to an ice-water bath and add 3.5 mL of thionyl chloride. Then stir at a speed of 300 rpm at room temperature for 12 h, add 1.5 g of potassium carbonate, mix evenly in an ice-water bath, add the oligomeric arginine-benzenesulfonamide prepared in step A4, and continue to stir and react at room temperature for 8 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain the polypeptide modifier;

[0068] This embodiment also provides a method for preparing an anti-tumor composition containing N-acetyl-D-glucosamine, which specifically includes the following steps:

[0069] ① Dissolve 1 g of functionalized N-acetyl-D-glucosamine and 3 g of polypeptide modifier by weight in a tert-butanol / water solution (volume ratio 2:1), add 0.1 g of sodium ascorbate and 0.2 g of copper sulfate pentahydrate, react with stirring at 80 °C for 2 h, then add ethyl acetate for extraction. After collecting the organic phase, dry it with anhydrous sodium sulfate, and after purification, obtain intermediate product B;

[0070] ② Dissolve the intermediate product B prepared in step ① in 10 mL of acetone, take 0.2 mL of a methanol solution of sodium methoxide with a concentration of 1 ml / L, react with stirring at room temperature for 30 min, then adjust the pH to neutral. Solid will precipitate out. Filter and collect the solid, and after purification, obtain the composition containing N-acetyl-D-glucosamine.

[0071] Figure 1 1H-NMR spectrum image of the compound in Example 1 of the present invention. Among them, A is functionalized N-acetyl-D-glucosamine, B is the polypeptide modifier, and C is the composition of N-acetyl-D-glucosamine. As shown in the figure, A: 1 1H-NMR(600MHz, DMSO-d 6 ) δ (ppm):8.05 (s, 1H, -NH),5.30 (d, 1H, H-1),4.90 (d, 1H, H-2),3.75-3.40 (m, 3H, H-3, H-4, H-5),2.05 (s, 3H, -CH 3 ).B: 1 1H-NMR (600 MHz, DMSO-d 6 ) δ (ppm):9.65 (s, 1H, -NH),8.15 (d, 2H, -H),7.65 (d, 2H, -H),7.00 (br, 1H,NH),5.25 (s, 1H, -CH),3.85 (m, 2H, -CH 2 -NH-),2.75 (t, 2H, -CH 2 -),2.05 (t, 2H,-CH 2 -),1.25 (s, 3H, -CH 3 ).C: 1 1H-NMR (600 MHz, DMSO-d 6) δ (ppm): 9.65 (s, 1H, -NH), 8.15 (d, 2H), 7.65 (d, 2H), 7.00 (br, 1H, NH), 6.65 (s, 1H, -H), 5.25 (d, 1H, H-1), 4.90 (d, 1H, H-2), 3.85 (m, 1H, H-3), 3.25 (m, 2H, H-4, H-5), 2.75 (t, 2H, -CH 2 -), 2.05 (t, 2H, CH 2 -), 1.25 (s, 3H, -CH 3 ).

[0072] Example 2

[0073] This example provides an anti-tumor composition containing N-acetyl-D-glucosamine, and the composition includes the following components in parts by weight: 2 parts of functionalized N-acetyl-D-glucosamine and 3 parts of polypeptide modifier;

[0074] The preparation method of functionalized N-acetyl-D-glucosamine specifically includes the following steps:

[0075] S1. Take 1 g of D-glucosamine and dissolve it in 20 mL of dichloromethane. Place it in an environment of 0 °C, add 1.1 mL of triethylamine, stir and mix for 10 min. Then take 1 mL and mix it evenly with 5 mL of dichloromethane to obtain a trichloroethyl chloroformate / dichloromethane solution. Slowly drip the trichloroethyl chloroformate / dichloromethane solution. While maintaining the condition of 0 °C, react for 30 - 50 min. After raising the reaction temperature to room temperature, continue to stir and react for 2 - 3 h. Adjust the reaction pH to neutral with saturated sodium bicarbonate solution, add dichloromethane for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain N-Troc glucosamine;

[0076] S2. Dissolve 0.5 g of the N-Troc-glucosamine prepared in step S1 in 25 mL of dichloromethane. Under the condition of an ice-water bath, mix hydrobromic acid and acetic acid according to a volume ratio of 1:2, drip the hydrobromic acid-acetic acid solution, stir at a speed of 120 rpm, react for 4 h, then add deionized water to quench the reaction. Neutralize the reaction system to neutral with saturated sodium bicarbonate aqueous solution, add dichloromethane for extraction, collect the organic phase, wash it with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain bromo-substituted-N-Troc-glucosamine;

[0077] S3. Take 0.5 g of bromo-substituted -N-Troc-glucosamine prepared in step S2, dissolve it in 50 mL of DMF, add 0.2 g of sodium azide and mix evenly. Stir at a speed of 200 rpm at room temperature. After reacting for 60 min, add water to quench the reaction. After adding ethyl acetate, collect the organic phase, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain azido-α-D-glucose;

[0078] S4. Dissolve 1 g of azido-α-D-glucose prepared in step S3 in 10 mL of anhydrous methanol, add 10 mL of glacial acetic acid, mix evenly, add 0.1 g of zinc powder, and react at 50 °C for 4 h. After filtration and collection of the filtrate, add 10 mL of acetic anhydride and stir and react at a speed of 150 rpm. After reacting for 3 h, add saturated sodium bicarbonate aqueous solution to adjust the pH to neutral, then add ethyl acetate for extraction, wash it with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify it to obtain functionalized N-acetyl-D-glucosamine;

[0079] The preparation raw materials of the polypeptide modifier include the following components in parts by weight: 20 parts of 4-aminobenzenesulfonamide, 4 parts of oligomeric arginine, and 30 parts of 4,5-hexynoic acid;

[0080] The preparation method of the polypeptide modifier specifically includes the following steps:

[0081] A1. Immerse the Rink Amide Resin in DCM and swell it for 30 min, then remove the solvent. Transfer the Rink Amide Resin to a 20 vol% pyridine / DMF solvent, stir and react for 10 min, then remove the pyridine / DMF solvent, add a new pyridine / DMF solvent and continue to stir and react for 10 min, and then wash the Rink Amide Resin with DMF to obtain the deprotected Rink Amide Resin;

[0082] A2. Weigh 1.2 g of Fmoc-Pbf-arginine and place it in a flask. Add 40 mL of DMF to fully dissolve Fmoc-Pbf-arginine. Then add 0.8 g of HBTU and 0.16 g of HOBt, and mix at a speed of 180 rpm. After mixing evenly, add 0.6 mL of DIEA, continue to mix for 5 min, and then add the Rink Amide Resin prepared in step A1. Place it at room temperature and stir the reaction at a speed of 300 rpm. After reacting for 1 h, filter to collect the solid Rink Amide Resin, and wash the Rink Amide Resin repeatedly with DMF to obtain Rink Amide Resin - oligomeric arginine;

[0083] A3. Accurately weigh 1 g of 4-carboxybenzenesulfonamide and place it in a flask. Add 20 mL of DMF to fully dissolve 4-carboxybenzenesulfonamide. Transfer the reaction system to an ice-water bath, add 4.0 mL of thionyl chloride, place it at room temperature, and stir at a speed of 200 rpm. After stirring and reacting overnight, use TLC to detect that the reaction is complete, then distill off the excess DMF under reduced pressure to obtain intermediate A. Add 30 mL of DMF to redissolve intermediate A, mix it with the Rink Amide Resin - oligomeric arginine in step A2, add triethanolamine to adjust the pH of the reaction system to 9.0, and react at 25 °C for 8 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain Rink Amide Resin - oligomeric arginine - benzenesulfonamide;

[0084] A4. Place the Rink Amide Resin - oligomeric arginine - benzenesulfonamide prepared in step A3 in 120 mL of TFA cleavage solution, stir and react at a speed of 150 rpm for 3 h, then filter to collect the filtrate. Add pre-cooled anhydrous ether for precipitation, centrifuge, discard the supernatant, collect the precipitate, and dry it to obtain oligomeric arginine - benzenesulfonamide;

[0085] A5. Accurately weigh 0.4 g of 4-pentynoic acid and dissolve it in 10 mL of dichloromethane. Transfer it to an ice-water bath and add 4.0 mL of thionyl chloride. Then stir at a speed of 200 rpm at room temperature for 16 h. Add 1.6 g of potassium carbonate, mix evenly in an ice-water bath, and then add the oligomeric arginine - benzenesulfonamide prepared in step A4. Continue to stir and react at room temperature for 10 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain the polypeptide modifier;

[0086] This embodiment also provides a method for preparing an anti-tumor composition containing N-acetyl-D-glucosamine, which specifically includes the following steps:

[0087] ① Dissolve 2 g of functionalized N-acetyl-D-glucosamine and 4 g of polypeptide modifier by weight in tert-butanol / water solution (volume ratio 3:1), add 0.4 g of sodium ascorbate and 0.6 g of copper sulfate pentahydrate, stir and react at 85 °C for 3 h, then add ethyl acetate for extraction. After collecting the organic phase, dry it with anhydrous sodium sulfate, and after purification, obtain intermediate product B;

[0088] ② Dissolve the intermediate product B prepared in step ① in 20 mL of acetone, take 0.25 mL of a methanol solution of sodium methoxide with a concentration of 1 ml / L, stir and react at room temperature for 40 min, then adjust the pH to neutral. Solid precipitates, filter, collect the solid, and after purification, obtain the composition containing N-acetyl-D-glucosamine.

[0089] Example 3

[0090] This embodiment provides an anti-tumor composition containing N-acetyl-D-glucosamine, and the composition includes the following components by weight: 3 parts of functionalized N-acetyl-D-glucosamine and 5 parts of polypeptide modifier;

[0091] The preparation method of functionalized N-acetyl-D-glucosamine specifically includes the following steps:

[0092] S1. Take 1.2 g of D-glucosamine and dissolve it in 20 mL of dichloromethane, place it in an environment of 0 °C, add 1.2 mL of triethylamine, stir and mix for 10 min, then take 0.9 mL and mix it evenly with 5 mL of dichloromethane to obtain a chloroformic acid-2,2,2-trichloroethyl ester / dichloromethane solution. Slowly drop the chloroformic acid-2,2,2-trichloroethyl ester / dichloromethane solution, keep the reaction at 0 °C for 30 - 50 min, raise the reaction temperature to room temperature, and continue to stir and react for 2 - 3 h. Adjust the reaction pH to neutral with saturated sodium bicarbonate solution, add dichloromethane for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain N-Troc glucosamine;

[0093] S2. Dissolve the N-Troc-glucosamine prepared in step S1 in 12.5 mL of dichloromethane. Under an ice-water bath condition, mix hydrobromic acid and acetic acid according to a volume ratio of 1:3, add the hydrobromic acid - acetic acid solution dropwise, stir at a speed of 180 rpm. After reacting for 2 h, add deionized water to quench the reaction. Neutralize the reaction system to neutral with saturated sodium bicarbonate aqueous solution, then add dichloromethane for extraction. Collect the organic phase, wash it with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, and then concentrate and purify to obtain bromo-substituted N-Troc-glucosamine;

[0094] S3. Take 0.5 g of the bromo-substituted N-Troc-glucosamine prepared in step S2 and dissolve it in 15 mL of DMF. Add 0.4 g of sodium azide and mix evenly. Stir at a speed of 300 rpm under room temperature condition. After reacting for 30 min, add water to quench the reaction. After adding ethyl acetate, collect the organic phase, wash it with saturated sodium chloride solution, dry the separated organic phase with anhydrous sodium sulfate, then filter, collect the filtrate, concentrate and purify to obtain azido-α-D-glucose;

[0095] S4. Dissolve 1 g of the azido-α-D-glucose prepared in step S3 in 20 mL of anhydrous methanol, add 15 mL of glacial acetic acid, mix evenly, then add 0.3 g of zinc powder, and react at 70 °C for 2 h. Filter, collect the filtrate, then add 15 mL of acetic anhydride, stir and react at a speed of 180 rpm. After reacting for 3 h, add saturated sodium bicarbonate aqueous solution to adjust the pH to neutral, then add ethyl acetate for extraction, wash it with saturated sodium chloride solution, dry the separated organic phase with anhydrous sodium sulfate, then filter, collect the filtrate, concentrate and purify to obtain functionalized N-acetyl-D-glucosamine;

[0096] A method for preparing a polypeptide modifier specifically includes the following steps:

[0097] A1. Immerse the Rink Amide Resin in DCM and swell it for 30 min, then remove the solvent. Transfer the Rink Amide Resin to a 25 vol% pyridine / DMF solvent, stir and react for 5 min, then remove the pyridine / DMF solvent, add a new pyridine / DMF solvent and continue to stir and react for 10 min, and then wash the Rink Amide Resin with DMF to obtain the deprotected Rink Amide Resin;

[0098] A2. Weigh 1.2 g of Fmoc-Pbf-arginine and place it in a flask. Add 30 mL of DMF to fully dissolve Fmoc-Pbf-arginine. Then add 0.67 g of HBTU and 0.4 g of HOBt, and mix at a speed of 150 rpm. After mixing evenly, add 0.4 mL of DIEA, continue to mix for 5 min, and then add the Rink Amide Resin prepared in step A1. Place it at room temperature and stir the reaction at a speed of 250 rpm. After reacting for 1.5 h, filter and collect the solid Rink Amide Resin. Wash the Rink Amide Resin repeatedly with DMF to obtain Rink Amide Resin - oligomeric arginine;

[0099] A3. Accurately weigh 0.9 g of 4-carboxybenzenesulfonamide and place it in a flask. Add 20 mL of DMF to fully dissolve 4-carboxybenzenesulfonamide. Transfer the reaction system to an ice-water bath, add 4.5 mL of thionyl chloride, place it at room temperature, and stir at a speed of 250 rpm. After stirring the reaction overnight, use TLC to detect that the reaction is complete, then distill off the excess DMF under reduced pressure to obtain intermediate A. Add 30 mL of DMF to redissolve intermediate A, mix it with the Rink Amide Resin - oligomeric arginine in step A2, add triethanolamine to adjust the pH of the reaction system to 8.0, and react at 25 °C for 8 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain Rink Amide Resin - oligomeric arginine - benzenesulfonamide;

[0100] A4. Place the Rink Amide Resin - oligomeric arginine - benzenesulfonamide prepared in step A3 in a 100 mL TFA cleavage solution and stir the reaction at a speed of 150 rpm for 2.5 h. Then filter and collect the filtrate. Add pre-cooled anhydrous ether for precipitation, centrifuge, discard the supernatant, collect the precipitate, and dry it to obtain oligomeric arginine - benzenesulfonamide;

[0101] A5. Accurately weigh 0.45 g of 4-pentynoic acid and dissolve it in 10 mL of dichloromethane. Transfer it to an ice-water bath and add 3.0 mL of thionyl chloride. Then stir at a speed of 250 rpm at room temperature for 14 h. Add 1.7 g of potassium carbonate, mix evenly in an ice-water bath, and then add the oligomeric arginine - benzenesulfonamide prepared in step A4. Continue to stir the reaction at room temperature for 12 h. Then add ethyl acetate for extraction, collect the organic phase, wash the organic phase with saturated sodium chloride solution, separate the organic phase, dry it with anhydrous sodium sulfate, filter, collect the filtrate, and concentrate and purify it to obtain the polypeptide modifier;

[0102] This embodiment also provides a method for preparing an anti-tumor composition containing N-acetyl-D-glucosamine, which specifically includes the following steps:

[0103] ① Dissolve 3 g of functionalized N-acetyl-D-glucosamine and 5 g of polypeptide modifier by weight in a tert-butanol / water solution (volume ratio of 2:1), add 0.9 g of sodium ascorbate and 0.9 g of copper sulfate pentahydrate, react with stirring at 90 °C for 2 h, then add ethyl acetate for extraction. After collecting the organic phase, dry it with anhydrous sodium sulfate, and after purification, obtain intermediate B;

[0104] ② Dissolve the intermediate B prepared in step ① in 15 mL of acetone, add 0.3 mL of a methanol solution of sodium methoxide with a concentration of 1 ml / L, stir and react at room temperature for 50 min, then adjust the pH to neutral. Solids will precipitate. Filter and collect the solids, and after purification, obtain the composition containing N-acetyl-D-glucosamine.

[0105] Comparative Example 1

[0106] This comparative example provides an anti-tumor composition and a method for preparing the same. The difference from Example 1 is only that steps A1 - A4 are not included, and p-carboxybenzenesulfonamide is directly reacted with 4-pentynoic acid to obtain the modifier, and the other components and their contents are the same as those in Example 1.

[0107] Comparative Example 2

[0108] This comparative example provides an anti-tumor composition and a method for preparing the same. The difference from Example 1 is only that steps S2 - S4 are not included, and the other components and their contents are the same as those in Example 1.

[0109] Experimental Example 1

[0110] In this experimental example, the carbonic anhydrase enzyme activity performance of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 was measured. Carbonic anhydrase can catalyze the hydrolysis of 4-nitrophenyl acetate (4-NPA) to produce 4-nitrophenol. In this experimental example, an absorbance photometer was used to measure the change in the absorbance value of 4-nitrophenol at 405 nm to measure the enzyme activity of carbonic anhydrase. The inhibition activity of the target compound on carbonic anhydrase was measured by the only method. The specific method includes: preparing a buffer solution with 15 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.01% tetraethylene glycol dodecyl ether, and 100 mmol / L sodium chloride, adjusting the pH of the buffer solution to 7.4, diluting carbonic anhydrase with the buffer solution to an enzyme concentration of 10 ng / μL, diluting 4-nitrophenyl acetate with the buffer solution to a substrate concentration of 1 nmol / L, preparing a 20 nmol / L stock solution of the positive control acetazolamide with DMSO, and preparing a 10 nmol / L stock solution of the test compound with DMSO. Take 18 μL of the CAIX solution into a 384-well reaction plate and centrifuge for standby; dilute acetazolamide and the test compound to different concentration gradients respectively, incubate the test compound with the enzyme at 25 °C for 15 min to form an enzyme-inhibitor complex, then add the substrate 4-NPA to the complex solution, incubate at 25 °C for 90 min, measure the absorbance value, and calculate the IC50 of the compound on carbonic anhydrase through the standard curve.

[0111] Figure 2 This is a graph showing the inhibitory activity results of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention on carbonic anhydrase. As shown in the figure, the IC50 of Examples 1-3 of the present invention is below 200 mmol / L, while the IC50 of the composition prepared in Comparative Example 1 is 540.5 mmol / L, and the IC50 of the composition prepared in Comparative Example 2 is 708.6 mmol / L. The above data show that the inhibitory activity of the compositions prepared in Examples 1-3 on carbonic anhydrase is obvious. In the present invention, oligolysine and sulfonyl groups with inhibitory activity are modified on the outside of NK cells. Arginine has more guanidyl groups, which can interact with the hydrophobic half of the active site, thereby producing an inhibitory effect on carbonic anhydrase.

[0112] Experimental Example 2

[0113] In this experimental example, the cell activities of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 were measured. Using 5-fluorouracil (5-FU) as a positive control drug, the in vitro anti-proliferation activity of the target compound against human colon cancer cells (HCT116) was tested by the MTT method. After the cells in logarithmic growth phase were digested with trypsin, the cell suspension concentration was adjusted to about 5×10 4 cells per milliliter with a culture medium containing fetal bovine serum (volume fraction of 10%), added 100 μL per well to a 96-well plate, and set a blank control with only the culture medium added, and placed it at 37 °C and a volume fraction of 5% CO2 Cultivate in an incubator for 24 h. In the experimental group, replace the culture medium with new culture media containing the test samples at different concentrations, while in the control group, replace the culture medium with a culture medium containing the same volume of solvent. Set up 3 parallel wells in each group and incubate at 37 °C and 5% CO 2 in the incubator for 48 h. Discard the supernatant, carefully wash twice with PBS, add 100 μL of MTT solution (5 mg·mL-1) to each well and continue to culture for 4 h. Discard the supernatant, add 100 μL of dimethyl sulfoxide to each well, place the 96-well plate on a micro shaker and shake for 3 min. After mixing, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the optical density (OD) value at 492 nm. Calculate the cell viability inhibition rate according to the negative control group. The formula is as follows, and the IC50 value of each compound is obtained by fitting a curve;

[0114]

[0115] Among them, : The average OD value of the blank control group; : The average OD value of the test group.

[0116] Figure 3 This is the inhibition activity result diagram of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention against HCT116. As shown in the figure, the IC50 of Examples 1-3 of the present invention is below 70 μmol / L, while the IC50 of the composition prepared in Comparative Example 1 is 234.5 μmol / L, and the IC50 of the composition prepared in Comparative Example 2 is 486.6 μmol / L. The above data show that the compositions prepared in Examples 1-3 have obvious inhibitory activity against carbonic anhydrase. In the present invention, oligolysine and sulfonyl groups with inhibitory activity are modified outside NK cells, and the nitrogen atoms of the sulfonamide groups all form coordination with Zn 2+ to form a coordination effect. The sulfonamide groups also form hydrogen bond interactions with Thr200 and Thr201 respectively. The oxygen atoms on the N-acetyl-D-glucosamine sugar ring all form hydrogen bond interactions with Thr73. The guanidine groups of oligolysine form hydrophobic interactions with Gln92. Therefore, the compositions described in the comparative examples cannot interact with the active center amino acids, resulting in a decrease in activity.

[0117] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

[0118] The above description of the present invention and its embodiments is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual application is not limited thereto. In general, if those of ordinary skill in the art are inspired by it and design, without creative efforts, ways and embodiments similar to the technical solution without departing from the spirit of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. An anti-tumor composition containing N-acetyl-D-glucosamine, characterized in that: The raw materials for preparing the composition include the following components in parts by weight: 1-3 parts of functionalized N-acetyl-D-glucosamine and 3-5 parts of polypeptide modification.

2. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 1, characterized in that: The preparation method of the functionalized N-acetyl-D-glucosamine specifically comprises the following steps: S1. Dissolve D-glucosamine in dichloromethane, place at 0°C, add triethylamine, stir and mix for 10 minutes, slowly drop 2,2,2-trichloroethyl chloroformate / dichloromethane solution, keep at 0°C, react for 30-50 minutes, raise the reaction temperature to room temperature, continue stirring and react for 2-3 hours, adjust the reaction pH to neutral with saturated sodium bicarbonate solution, add dichloromethane for extraction, wash the organic phase with saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, concentrate and purify to obtain N-Troc glucosamine; S2, dissolving the N-Troc-glucosamine prepared in step S1 in dichloromethane, adding hydrobromic acid-acetic acid solution dropwise in an ice-water bath, stirring at a speed of 120-180 rpm, reacting for 2-4 hours, adding deionized water to quench the reaction, neutralizing the reaction system with a saturated sodium bicarbonate aqueous solution to neutrality, adding dichloromethane for extraction, collecting the organic phase, washing with a saturated sodium chloride aqueous solution, drying the organic phase with anhydrous sodium sulfate, concentrating and purifying to obtain bromine-substituted-N-Troc-glucosamine; S3, dissolving the bromine-substituted-N-Troc-glucosamine prepared in step S2 in DMF, adding sodium azide to mix evenly, stirring at 200-300 rpm at room temperature, reacting for 30-60 min, adding water to quench the reaction, adding ethyl acetate, collecting the organic phase, dissolving it with saturated sodium chloride for washing, separating the organic phase, drying it with anhydrous sodium sulfate, filtering, collecting the filtrate, concentrating and purifying it to obtain azidized α-D-glucose; S4. Dissolve the azidated α-D-glucose prepared in step S3 in anhydrous methanol, add glacial acetic acid, mix well, add zinc powder, react at 50-70°C for 2-4h, filter, collect the filtrate, transfer to an ice-water bath, add acetic anhydride, stir and react at a speed of 150-180rpm, react for 2-3h, add saturated sodium bicarbonate aqueous solution to adjust the pH to neutral, add ethyl acetate for extraction, dissolve and wash with saturated sodium chloride, separate the organic phase, dry with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify, and functionalize N-acetyl-D-glucosamine.

3. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 2, characterized in that: In step S1, the weight concentration of D-glucosamine in dichloromethane is 40-60 mg / mL; The mass volume ratio between the D-glucosamine and triethylamine is 0.7-0.9 g / mL; The mass volume ratio between the D-glucosamine and 2,2,2-trichloroethyl chloroformate is 1-1.2 g / mL.

4. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 3, characterized in that: In step S2, the mass concentration of N-Troc-glucosamine in dichloromethane is 20-40 mg / mL; The volume ratio of hydrobromic acid to acetic acid in the hydrobromic acid-acetic acid solution is 1:2-3; The mass volume ratio between the N-Troc-glucosamine and the hydrobromic acid-acetic acid solution is 0.05-0.15 g / mL.

5. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 4, characterized in that: In step S3, the mass concentration of the bromine-substituted-N-Troc-glucosamine in DMF is 10-30 mg / mL; The mass ratio of the bromine-substituted-N-Troc-glucosamine to the obtained sodium iodide is 5:2-4.

6. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 5, characterized in that: In step S4, the mass concentration of the azidated α-D-glucose in anhydrous methanol is 0.05-0.1 g / mL; The volume ratio between the glacial acetic acid and anhydrous methanol is 0.75-1.2:1; The added mass of zinc is 10-30% of the mass of azid α-D-glucose; The mass volume ratio between the azidated α-D-glucose and acetic anhydride is 10-50 mg / mL.

7. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 6, characterized in that: The preparation method of the modified polypeptide specifically comprises the following steps: A1. Soak the Rink Amide Resin in DCM to swell it, remove the solvent, transfer the Rink Amide Resin to a pyridine / DMF solution, stir and react for 5-10 minutes, remove the pyridine / DMF solvent, add new pyridine / DMF solvent, continue stirring and reacting for 10-15 minutes, and wash the Rink Amide Resin with DMF to obtain a deprotected Rink Amide Resin. A2, dissolving Fmoc-Pbf-arginine in DMF, adding HBTU and HOBt, mixing thoroughly, adding DIEA, stirring at 150-180 rpm for 3-5 min, adding the deprotected Rink Amide Resin prepared in step A1, stirring at 200-300 rpm for 1-2 h at room temperature, filtering, collecting the Rink Amide Resin, and repeatedly washing with DMF to obtain Rink Amide Resin-oligoarginine; A3, dissolving p-carboxybenzenesulfonamide in DMF, adding dichlorothionyl dropwise in an ice-water bath, stirring at 200-300 rpm overnight at room temperature, detecting the reaction completion by TLC, removing the solvent to obtain intermediate product A, dissolving intermediate product A in DMF, adding to the reaction system in step A2, adding TEA to keep the pH of the reaction system at 8-9, reacting at 25-40° C. for 4-8 hours, adding ethyl acetate for extraction, collecting the organic phase, washing with a saturated sodium chloride solution, separating the organic phase, drying with anhydrous sodium sulfate, filtering, collecting the filtrate, concentrating and purifying to obtain Rink Amide Resin-oligoarginine-benzenesulfonamide; A4, placing the Rink Amide Resin-oligoarginine-benzenesulfonamide prepared in step A3 in a TFA cutting solution, stirring at 150-180 rpm for 2-3 hours, filtering, collecting the filtrate, adding pre-cooled anhydrous ether for precipitation, centrifuging, discarding the supernatant, collecting the precipitate, and drying to obtain oligoarginine-intermediate product A; A5. Dissolve 4-pentynoic acid in dichloromethane, place in an ice water bath, add dichlorothionyl, raise the temperature to room temperature, stir at 200-300 rpm for 12-16 hours, add potassium carbonate, transfer to ice water, add oligoarginine-benzenesulfonamide prepared in step A4, stir at 200-300 rpm for 8-12 hours, add ethyl acetate for extraction, collect the organic phase, wash with saturated sodium chloride solution, separate the organic phase, dry with anhydrous sodium sulfate, filter, collect the filtrate, concentrate and purify to obtain a modified polypeptide.

8. The anti-tumor composition containing N-acetyl-D-glucosamine according to claim 7, characterized in that: In step A1, the volume fraction of pyridine in DMF in the pyridine / DMF solution is 15%-25%; In step A2, the mass concentration of Fmoc-Pbf-arginine in DMF is 30-50 mg / mL In step A2, the mass ratio of Fmoc-Pbf-arginine, HBTU and HOBt is 1.5-2:1:0.2-0.6; In step A2, the mass volume ratio between Fmoc-Pbf-arginine and DIEA is 2.0-2.5 g / mL In step A3, the mass concentration of p-carboxybenzenesulfonamide in DMF is 40-50 mg / mL; In step A3, the mass volume ratio between p-carboxybenzenesulfonamide and dithionyl chloride is 0.2-0.25 g / mL; In step A4, the volume of the TFA cleavage solution is 100-150 mL; In step A5, the mass concentration of 4-pentynoic acid in dichloromethane is 40-50 mg / mL; In step A5, the mass volume ratio between the 4-pentynoic acid and thionyl chloride is 0.1-0.15 g / mL: In step A5, the mass ratio between 4-pentynoic acid and potassium carbonate is 1:2.5-4.

9. A method for preparing the anti-tumor N-acetyl-D-glucosamine-containing composition according to any one of claims 1 to 8, characterized in that: The specific steps include: ① Dissolve the functionalized N-acetyl-D-glucosamine and the peptide modification in tert-butyl alcohol / water solution according to weight parts, add sodium ascorbate and copper sulfate pentahydrate, react at 80-90° C., stir for 2-3 hours, add ethyl acetate for extraction, collect the organic phase, dry it with anhydrous sodium sulfate, and purify it to obtain intermediate product B; ② The intermediate product B prepared in step ① is dissolved in acetone, and a methanol solution of sodium methoxide is added. After stirring and reacting for 30-50 minutes at room temperature, the pH is adjusted to neutral, and solids are precipitated. The solids are filtered and collected. After purification, a composition containing N-acetyl-D-glucosamine is obtained.

10. The method for preparing an anti-tumor composition containing N-acetyl-D-glucosamine according to claim 9, characterized in that: In step ①, in the tert-butyl alcohol / water solution, the mass ratio between tert-butyl alcohol and deionized water is 2-3:1; In step ①, the mass ratio of the energy-soluble N-acetyl-D-glucosamine, sodium ascorbate and copper sulfate pentahydrate is 10:1-3:2-3; In step ②, the volume of acetone is 10-20 mL; In step ②, the amount of sodium methoxide added is 10-16 mg.

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