Preparation method of targeted drug polypeptide for treating glioma as well as product and application of targeted drug polypeptide

Abstract

The invention discloses a preparation method of a targeted drug polypeptide for glioma treatment, which comprises the following steps: 1) preparing a small molecule inhibitor couple TGN020-5ca of AQP4, and the structural formula of the TGN020-5ca is as follows: 2) sequentially adding different amino acids into a solid-phase synthesis tube to obtain a polypeptide, and reacting the polypeptide with trimethylbromosilane and methanol to obtain a methyl esterified polypeptide; and 3) preparing the polypeptide-TGN020: firstly adding N-hydroxysuccinimide and N, N '-diisopropyl carbodiimide, activating carboxyl on the TGN020-5ca at the activation temperature of 37-60 DEG C for 6-8 hours, then adding polypeptide or methyl esterified polypeptide and N, N-diisopropyl ethylamine, and reacting at the temperature of 25-37 DEG C for 1-2 hours, the molar ratio of the TGN020-5ca to the polypeptide or methyl esterified polypeptide being 1.2: 1-1.5: 1. The polypeptide TGN020 is targeted and accumulated on a glioma cell membrane, can inhibit the rate of water molecules entering and exiting from glioma cells in a long-acting manner, effectively inhibits the malignant proliferation rate of glioma, has the capabilities of targeted enrichment and long-acting AQP4 inhibition, and has excellent glioma cell killing capability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a targeted drug polypeptide for glioma treatment, a product thereof and application. BACKGROUND

[0002] Glioma is the most common and most invasive primary intracranial tumor, among which the malignant degree of glioblastoma multiforme (GBM) is the highest, and the prognosis of patients is extremely poor. The current standard treatment methods (including surgery, radiotherapy and chemotherapy) have limited effect and high recurrence rate, so it is urgent to develop new targeted treatment methods. Aquaporin 4 (AQP4) is specifically highly expressed in glioma cells, and is closely related to the migration and proliferation of tumor cells and the formation of brain edema. Inhibition of the function of AQP4 has been proved to effectively inhibit the growth of glioma, so AQP4 has become a very potential therapeutic target. However, the existing AQP4 small molecule inhibitors or antibody drugs have problems such as low affinity, poor specificity, fast metabolism in vivo, difficulty in penetrating the blood-brain barrier (BBB) and insufficient enrichment in the tumor site, which seriously limit their clinical transformation and application. Tumor cells usually exhibit different enzyme levels than normal cells, so enzyme-triggered supramolecular self-assembly based on small molecule polypeptides often exhibit excellent selectivity for tumor cells. In recent years, such materials have shown great application prospects in drug controlled release, cancer diagnosis and treatment, regenerative medicine and the like. Supramolecular assemblies generated by self-assembly of small molecule polypeptides through non-covalent bonds have unique advantages: simple synthesis, easy functionalization and clear functional units, good biocompatibility, low immunogenicity and low toxicity, inherent biodegradability and rapid stimulation responsiveness to the external environment, etc. The present application is based on the highly active alkaline phosphatase (ALP) on the membrane surface of glioma cells, which constructs a nanostructure in situ on the membrane surface of glioma cells to multivalently bind and inhibit AQP4, thereby realizing high-efficiency anti-tumor effect. SUMMARY

[0003] Therefore, the present application provides a preparation method of a targeted drug polypeptide for glioma treatment, a product thereof and application.

[0004] The present application specifically provides the following technical solutions:

[0005] 1. A preparation method of a targeted drug polypeptide for glioma treatment, the preparation steps being:

[0006] 1) preparing a small molecule inhibitor TGN020-5ca of AQP4, wherein the structure of the TGN020-5ca is

[0007] 2) Preparation of polypeptide or methyl esterified polypeptide: different amino acids are added to a solid phase synthesis tube in sequence to obtain a polypeptide, and the polypeptide is reacted with trimethylsilyl bromide and methanol to obtain a methyl esterified polypeptide;

[0008] 3) Coupling of a small molecule inhibitor of AQP4 to a polypeptide molecule to prepare a polypeptide-TGN020: N-hydroxysuccinimide and N,N'-diisopropylcarbodiimide are first added to activate the carboxyl group on TGN020-5ca, the activation temperature is 37-60°C, and the activation time is 6-8 hours, then the polypeptide or methyl esterified polypeptide and N,N-diisopropylethylamine are added, the reaction temperature is 25-37°C, and the reaction time is 1-2 hours, and the molar ratio of TGN020-5ca to the polypeptide or methyl esterified polypeptide is 1.2:1-1.5:1.

[0009] Further, in step 3), the amount of N-hydroxysuccinimide added is 1.5 equivalents, the amount of N,N'-diisopropylcarbodiimide added is 3 equivalents, and the amount of N,N-diisopropylethylamine added is 3 equivalents, based on the amount of substance of the polypeptide or methyl esterified polypeptide.

[0010] Further, the sequence of the polypeptide of step 2) is NapFFKYp or NapKYpF or NBD-AFFKYp or NBD-AFFYpKK.

[0011] Further, step 1) is specifically:

[0012] 1) Preparation of 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylic acid ethyl ester nicotinic acid;

[0013] 2) Preparation of 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylic acid ethyl ester from 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylic acid ethyl ester nicotinic acid of step 1);

[0014] 3) Preparation of 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid from 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylic acid ethyl ester of step 2);

[0015] 4) Dissolution of 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid of step 3) in acetonitrile, addition of 2-amino-1,3,4-thiadiazole, N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate and N-methylimidazole, stirring at room temperature for 8-12 hours, rotary evaporation of the obtained mixture to obtain a solid, extraction with ethyl acetate and saturated brine, adjustment of pH to 2-4 with HCl, collection of the organic phase, rotary evaporation, and purification of the solid residue by silica gel column chromatography to obtain N-(2-amino-1,3-thiazol-5-yl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide;

[0016] 4) Dissolve N-(2-amino-l,3-thiazol-5-yl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide of step 4) in water, add trifluoroacetic acid and methanol, stir at room temperature for 6-8 h, remove the solvent by rotary evaporation to obtain a white solid, dissolve in DMF, add glutaric anhydride and N,N-diisopropylethylamine, stir the mixture at room temperature for 6-8 h, elute and purify, and freeze-dry to obtain TGN020-5ca.

[0017] Further, when the polypeptide sequence of step 2) is NapFFKYp, the preparation method is as follows: swell 2-chloro-trityl chloride resin in super dry dichloromethane, dissolve Fmoc-O-benzyl-L-phosphotyrosine and N,N-diisopropylethylamine in DMF and add to the resin, react at room temperature for 6-12 h, filter, wash, add methanol / dichloromethane (v / v = 1:4) again, react at room temperature for 30 min, filter, wash with DMF, remove Fmoc protection with 20% piperidine, then add L-lysine, L-phenylalanine, L-phenylalanine, 2-naphthaleneacetic acid in the ratio of resin: amino acid: HBTU: DIPEA = 1:3:3:6 in sequence, wash at each step and remove Fmoc protection with 20% piperidine, finally cut the resin with trifluoroacetic acid and precipitate with ice ethyl ether, purify by HPLC, and freeze-dry to obtain the polypeptide molecule.

[0018] Further, when the polypeptide sequence of step 2) is NapKYpF, the preparation method is as follows: swell 2-chloro-trityl chloride resin in super dry dichloromethane, dissolve Fmoc-L-phenylalanine and N,N-diisopropylethylamine in DMF and add to the resin, react at room temperature for 6-12 h, filter, wash, add methanol / dichloromethane (v / v = 1:4) again, react at room temperature for 30 min, filter, wash with DMF, remove Fmoc protection with 20% piperidine, then add Fmoc-O-benzyl-L-phosphotyrosine, L-lysine, 2-naphthaleneacetic acid in the ratio of resin: amino acid: HBTU: DIPEA = 1:3:3:6 in sequence, wash at each step and remove Fmoc protection with 20% piperidine, finally cut the resin with trifluoroacetic acid and precipitate with ice ethyl ether, purify by HPLC, and freeze-dry to obtain the polypeptide molecule.

[0019] Further, when the polypeptide sequence of step 2) is NBD-AFFKYp, the preparation method is as follows: 2-chloro-trityl chloride resin is swelled in super dry dichloromethane, Fmoc-O-benzyl-L-phosphotyrosine and N,N-diisopropyl ethylamine are dissolved in DMF and added to the resin, and the reaction is carried out at room temperature for 6-12 hours, then filtered, washed, and then added with methanol / dichloromethane, and the reaction is carried out at room temperature for 30 minutes, then filtered, washed with DMF, and then deprotected by 20% piperidine, then L-lysine, L-phenylalanine, L-phenylalanine, NBD-β-alanine are sequentially added in the proportion of resin:amino acid:HBTU:DIPEA=1:3:3:6, and washed at each step and deprotected by 20% piperidine, and finally the resin is cut by trifluoroacetic acid and precipitated by ice ethyl ether, purified by HPLC, freeze-dried, and the polypeptide molecule is obtained.

[0020] Further, when the polypeptide sequence of step 2) is NBD-AFFKYp, the preparation method is as follows: 2-chloro-trityl chloride resin is swelled in super dry dichloromethane, Fmoc-O-benzyl-L-phosphotyrosine and N,N-diisopropyl ethylamine are dissolved in DMF and added to the resin, and the reaction is carried out at room temperature for 6-12 hours, then filtered, washed, and then added with methanol / dichloromethane, and the reaction is carried out at room temperature for 30 minutes, then filtered, washed with DMF, and then deprotected by 20% piperidine, then L-lysine, Fmoc-O-benzyl-L-phosphotyrosine, L-phenylalanine, L-phenylalanine, NBD-β-alanine are sequentially added in the proportion of resin:amino acid:HBTU:DIPEA=1:3:3:6, and washed at each step and deprotected by 20% piperidine, and finally the resin is cut by trifluoroacetic acid and precipitated by ice ethyl ether, purified by HPLC, freeze-dried, and the polypeptide molecule is obtained.

[0021] 2. The preparation method of the targeted drug polypeptide for glioma treatment.

[0022] 3. The application of the targeted drug polypeptide for glioma treatment as a sensitizing drug of a chemotherapy drug for inhibiting glioma cells, characterized by being used for inhibiting aquaporin 4 and the inhibition time being greater than or equal to 72 hours.

[0023] The beneficial effects of the present application are as follows:

[0024] 1. Based on the small molecule inhibitor (TGN020) of aquaporin 4 (AQP4), the pyridine ring No. 5 of TGN020 is functionalized, and the TGN020 molecular derivative TGN020-5ca at No. 5 is synthesized and prepared, and the derivative molecule can covalently connect other chemical structure molecules by using the terminal carboxyl group.

[0025] 2. The polypeptide-TGN020 with TGN020 ligand obtained by covalently connecting TGN020 and polypeptide with alkaline phosphatase response and self-assembly function through NHS amidation reaction method can be enriched on the surface of glioma U87MG cell membrane, realizes the targeting function of U87MG cell membrane, and realizes the technical effects of polypeptide assembly targeting and accumulating on the glioma cell membrane.

[0026] 3. The polypeptide-TGN020 of the present application targets and accumulates on the glioma cell membrane, can long-acting inhibit (≥72 hours) the rate of water molecules entering and exiting the glioma cell, and effectively inhibits the malignant proliferation rate of glioma, has the ability of targeting enrichment and long-acting inhibition of AQP4, and has excellent glioma cell killing ability.

[0027] 4. The polypeptide-TGN020 of the present application can effectively perform chemosensitization of chemotherapeutic drug temozolomide by inhibiting the rate of water molecules entering and exiting mediated by AQP4 protein, and synergistically kill and inhibit glioma. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings:

[0029] Figure 1 The ESI-MS spectrum of NapFFKYp-OMe-TGN020. 1 H-NMR spectrum.

[0030] Figure 2 The ESI-MS spectrum of NapFFKYp-OMe-TGN020.

[0031] Figure 3 The morphology diagram of polypeptide-TGN020 on the surface of glioma cell U87MG cell membrane taken by scanning electron microscope.

[0032] Figure 4 The assembly distribution diagram of polypeptide-TGN020 on the glioma cell U87MG cell membrane taken by laser confocal microscope.

[0033] Figure 5 The rate of water molecules entering and exiting AQP4 on the U87MG cell membrane detected by magnetic resonance imaging method.

[0034] Figure 6 Toxicity test of polypeptide-TGN020 on glioma cell U87MG.

[0035] Figure 7 Tumor volume change diagram of polypeptide-TGN020 combined with temozolomide for ectopic glioma.

[0036] Figure 8TGN020-5ca 1 H-NMR spectrum. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present application will be described in detail by referring to the attached drawings.

[0038] Example 1 Preparation of TGN020-5ca

[0039] (1) 250 mg (1.09 mmol) of ethyl 5-bromonicotinate, 38 mg (0.0545 mmol) of bis(triphenylphosphine)palladium dichloride, 423 mg (1.31 mmol) of N,N- diisopropylethylamine (DIPEA) were dissolved in 5 mL of super-dry acetonitrile under N2protection, and 203 mg (1.31 mmol) of N-Boc-aminopropynyl and 15 mg (0.0763 mmol) of cuprous iodide were added to the solvent system. After the addition was completed, the reaction mixture was heated to reflux at 80°C for 6 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, 50 mL of water and 50 mL of dichloromethane were added, the pH was adjusted to 2-4 with 1 mol / L hydrochloric acid, and extraction was performed. The organic phase was collected and dried over anhydrous sodium sulfate, and the resulting mixture was purified by silica gel column chromatography (0-33%, ethyl acetate-n-hexane). The resulting yellow oil was frozen at -20°C for 16 h to obtain a yellowish white solid, ethyl 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylate (yield: 61%).

[0040] (1.31 mmol) of N,N-diisopropylethylamine (DIPEA) were dissolved in 5 mL of super-dry acetonitrile under N2protection, and 203 mg (1.31 mmol) of N-Boc-aminopropynyl and 15 mg (0.0763 mmol) of cuprous iodide were added to the solvent system. After the addition was completed, the reaction mixture was heated to reflux at 80°C for 6 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, 50 mL of water and 50 mL of dichloromethane were added, the pH was adjusted to 2-4 with 1 mol / L hydrochloric acid, and extraction was performed. The organic phase was collected and dried over anhydrous sodium sulfate, and the resulting mixture was purified by silica gel column chromatography (0-33%, ethyl acetate-n-hexane). The resulting yellow oil was frozen at -20°C for 16 h to obtain a yellowish white solid, ethyl 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylate (yield: 61%).

[0041] Ethyl pyridine-2-carboxylate nicotinate (yield: 61%).

[0042] (2) 100 mg (0.346 mmol) of ethyl 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylate nicotinate was dissolved in 5 mL of methanol and transferred to a three-necked flask, and the flask was replaced with N2. After cooling in ice water, 20 mg (20% w / w) of Pd / C was added and stirred, and a balloon device for H2was connected to make it an H2atmosphere. The reaction was stirred for 6 h. After completion, the mixture was filtered through celite. The solvent was removed by rotary evaporation, and extraction was performed with water and dichloromethane. The organic phase was collected and rotary evaporated. The resulting residue was purified by silica gel column chromatography (0-50%, ethyl acetate-n-hexane), and rotary evaporation was performed to obtain a yellowish white solid, ethyl 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylate (yield: 98%).

[0043] (2) 100 mg (0.346 mmol) of ethyl 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylate nicotinate was dissolved in 5 mL of methanol and transferred to a three-necked flask, and the flask was replaced with N2. After cooling in ice water, 20 mg (20% w / w) of Pd / C was added and stirred, and a balloon device for H2was connected to make it an H2atmosphere. The reaction was stirred for 6 h. After completion, the mixture was filtered through celite. The solvent was removed by rotary evaporation, and extraction was performed with water and dichloromethane. The organic phase was collected and rotary evaporated. The resulting residue was purified by silica gel column chromatography (0-50%, ethyl acetate-n-hexane), and rotary evaporation was performed to obtain a yellowish white solid, ethyl 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylate (yield: 98%).

[0044] (3) 100 mg (0.324 mmol) of 4-[3-(isopropoxycarbonylamino)propyl]pyridine-2- carboxylic acid ethyl ester was dissolved in 2 mL of methanol and 4 mol / L NaOH was added, and the reaction was stirred at 37°C for 4 h. After rotary evaporation, the pH was adjusted to 2-4 with 1 mol / L HCl, and the mixture was lyophilized to remove the solvent. The solid residue was purified by silica gel column chromatography (90:9:1 dichloromethane-methanol-acetic acid) to obtain 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid as a yellowish white solid (yield: 90%).

[0045] (4) 55 mg (0.179 mmol) of 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid was dissolved in 2 mL of acetonitrile, and 23.84 mg (0.214 mmol) of 2-amino-1,3,4-thiadiazole, 58 mg (0.188 mmol) of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate, and 55 μL (0.5265 mmol) of N-methylimidazole were added. The reaction was stirred at room temperature for 12 h. The resulting mixture was rotary evaporated to obtain a solid, which was extracted with ethyl acetate and saturated brine, the pH was adjusted to 2-4 with 1 mol / L HCl, and the organic phase was collected and rotary evaporated. The solid residue was purified by silica gel column chromatography (0-10% methanol-dichloromethane) to obtain N-(2-amino-1,3-thiazol-5-yl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide as a white solid (yield: 38%).

[0046]

[0047] (5) 100 mg (0.275 mmol) of N-(2-amino-1,3-thiazol-5-yl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide was dissolved in 1 mL of water, 1 mL of trifluoroacetic acid and 1 mL of methanol were added, and the mixture was stirred at room temperature for 8 h. The solvent was removed by rotary evaporation to obtain a white solid. The solid was dissolved in 2 mL of DMF, 65 mg (0.57 mmol) of glutaric anhydride and 120 μL (1.14 mmol) of N,N-diisopropylethylamine (DIPEA) were added, and the mixture was stirred at room temperature for 8 h. The sample was then injected into a semi-preparative high-performance liquid chromatography column, and purified using a water-acetonitrile gradient elution. The resulting solvent was lyophilized to obtain TGN020-5ca as a white powder (yield: 60%).

[0048] (1.14 mmol) N,N-diisopropylethylamine (DIPEA), and the mixture was stirred at room temperature for 8 h. The sample was then injected into a semi-preparative high-performance liquid chromatography column, and purified using a water-acetonitrile gradient elution. The resulting solvent was lyophilized to obtain TGN020-5ca as a white powder (yield: 60%).

[0049] The structure of TGN020-5ca is as follows:

[0050]

[0051] TGN020-5ca thus prepared was subjected to the following tests 1 ​H-NMR test characterization, Figure 8 TGN020-5ca 1 H-NMR spectrum. The assignment of the Figure 8 H-NMR spectrum. The assignment of the 1 H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 9.08 (s, 1H), 8.72 (s, 1H), 8.39 (t, J = 2.0 Hz, 1H), 7.89 (t, J = 5.6 Hz, 1H), 3.07 (t, J = 6.4 Hz, 2H), 2.71 (t, J = 7.7 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 2.10 (t, J = 7.4 Hz, 2H), 1.83 - 1.64 (m, 4H)). The above can prove that TGN020-5ca is successfully obtained. The 5th position of the pyridine ring of TGN020 is successfully functionalized.

[0052] Example 2 Preparation of polypeptide (NapFFKYp)

[0053] (1) In a solid-phase synthesis tube, 1.0 g of 2-chloro-trityl resin was swelled in 10 mL of super dry dichloromethane for 30 min. 1.3 g (3.0 mmol) of Fmoc-0-benzyl-L- phosphotyrosine and 992 μL (6.0 mmol) of N,N-diisopropylethylamine (DIPEA) were dissolved in 5 mL of super dry DMF and added to the resin, reacted at room temperature for 12 h, filtered,

[0054] DMF washing.

[0055] (2) 10 mL of methanol / dichloromethane (v / v = 1:4) was added to the resin, reacted at room temperature for 30 min, filtered, and DMF washed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, reacted at room temperature for 30 min, filtered, and DMF washed.

[0056] (3) 1.4 g (3.0 mmol) of Fmoc-tert-butoxycarbonyl-L-lysine (K), 1.14 g (3.0 mmol) of benzotriazole

[0057] -N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, reacted at room temperature for 2 h. Filtered, DMF washed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, reacted at room temperature for 30 min. Filtered, DMF washed.

[0058] (4) 1.2 g (3.0 mmol) of Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) of HBTU and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, and reacted at room temperature for 2 h. Filtration and DMF washing were performed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, and reacted at room temperature for 30 min. Filtration and DMF washing were performed.

[0059] DIPEA was dissolved in 5 mL of DMF and added to the resin, and reacted at room temperature for 2 h. Filtration and DMF washing were performed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, and reacted at room temperature for 30 min. Filtration and DMF washing were performed.

[0060] (5) 1.2 g (3.0 mmol) of Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) of HBTU and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, and reacted at room temperature for 2 h. Filtration and DMF washing were performed.

[0061] DIPEA was dissolved in 5 mL of DMF and added to the resin, and reacted at room temperature for 2 h. Filtration and DMF washing were performed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, and reacted at room temperature for 30 min. Filtration and DMF washing were performed.

[0062] (6) 0.56 g (3.0 mmol) of 2-naphthaleneacetic acid (Nap), 1.14 g (3.0 mmol) of HBTU and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, and reacted at room temperature for 2 h under nitrogen gas. Filtration and DMF washing were performed.

[0063] (7) 5 mL of trifluoroacetic acid was added to the resin, and reacted at room temperature for 2 h. Filtration was performed to obtain a filtrate. The filtrate was added dropwise to 30 mL of ice-ethanol, and filtration was performed to obtain a white solid. The obtained crude product was purified using semi-preparative HPLC to obtain a polypeptide (NapFFKYp), and the yield was 60%.

[0064] Example 3 Preparation of methyl esterified polypeptide (NapFFKYp-OMe)

[0065] (1) 32 mg (0.037 mmol) of NapFFKYp was dissolved in 2 mL of super-dry dichloromethane. 101.28 μL (0.74

[0066] mmol) of trimethylsilyl bromide was added dropwise, and stirred at room temperature for 12 h.

[0067] (2) The reaction solution was evaporated to remove the solvent, and 2 mL of super-dry methanol was added to the residue and stirred again for 12 h. After the reaction was completed, 1 mL of water was added to quench the reaction.

[0068] (3) Evaporation was performed to obtain a solid, which was dissolved in water and acetonitrile. The obtained crude product was purified using semi-preparative HPLC to obtain a polypeptide derivative (NapFFKYp-OMe), and the yield was 90%.

[0069] Example 4 Preparation of polypeptide-TGN020 series material NapFFKYp-OMe-TGN020

[0070] (1) 32 mg (0.0849 mmol) TGN020-5ca, 9.67 mg (1.02 mmol) N-hydroxysuccinimide, 13 μL (1.02

[0071] mmol) N,N'-diisopropylcarbodiimide (DIC) were dissolved in 1 mL DMF, and heated and stirred at 37 °C for 8 h.

[0072] (2) After the reaction was completed, 0.5 mL water was added to quench the DIC, and 61.2 mg (0.07 mmol) NapFFKYp-OMe and 23 μL (0.14 mmol) N,N-diisopropylethylamine (DIPEA) were added to the system, which was heated and stirred at 37 °C for 2 h. The resulting mixture was purified by semi-preparative HPLC to obtain the final product (NapFFKYp-OMe-TGN020), with a yield of 56%.

[0073] The structural formula of NapFFKYp-OMe-TGN020 is:

[0074]

[0075] Example 5 Preparation of polypeptide-TGN020 series material NapFFKYp-TGN020

[0076] (1) 32 g (0.0849 mmol) TGN020-5ca, 9.67 mg (1.02 mmol) N-hydroxysuccinimide, 13 mL (1.02

[0077] mmol) N,N'-diisopropylcarbodiimide (DIC) were dissolved in 1 mL DMF, and heated and stirred at 37 °C for 8 h.

[0078] (2) After the reaction was completed, 0.5 mL water was added to quench the DIC. 61.1 mg (0.07 mmol) NapFFKYp and 23 mL

[0079] (0.14 mmol) DIPEA were added to the system, which was heated and stirred at 37 °C for 2 h. The resulting mixture was purified by semi-preparative HPLC to obtain the final product (NapFFKYp-TGN020), with a yield of 60%.

[0080] The structural formula of NapFFKYp-TGN020 is:

[0081]

[0082] Example 6 Preparation of polypeptide - NapKYpF-TGN020 series of materials

[0083] (1) In a solid phase synthesis tube, 1.0 g of 2-chloro-trityl resin was swelled in 10 mL of super dry dichloromethane for 30 min. 1.2 g (3.0 mmol) of Fmoc-L-phenylalanine (F) and 992 μL (6.0 mmol) of N,N-diisopropylethylamine (DIPEA) were dissolved in 5 mL of super dry DMF and added to the resin, reacted at room temperature for 12 h, filtered, and washed with DMF.

[0084] (2) 10 mL of methanol / dichloromethane (v / v = 1 :4) was added to the resin, reacted at room temperature for 30 min, filtered, and washed with DMF. 10 mL of piperidine / DMF (v / v = 1 :4) was added to the resin, reacted at room temperature for 30 min, filtered, and washed with DMF.

[0085] (3) 1.3 g (3.0 mmol) of Fmoc-0-benzyl-L-phosphotyrosine (Yp), 1.14 g (3.0 mmol) of benzotriazole

[0086] -N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, reacted at room temperature for 2 h. Filtered, and washed with DMF. 10 mL of piperidine / DMF (v / v = 1 :4) was added to the resin, reacted at room temperature for 30 min. Filtered, and washed with DMF.

[0087] (4) 1.4 g (3.0 mmol) of Fmoc-L-lysine (K), 1.14 g (3.0 mmol) of HBTU, and 992 μL (6.0 mmol)

[0088] DIPEA were dissolved in 5 mL of DMF and added to the resin, reacted at room temperature for 2 h. Filtered, and washed with DMF. 10 mL of piperidine / DMF (v / v = 1 :4) was added to the resin, reacted at room temperature for 30 min. Filtered, and washed with DMF.

[0089] (5) 0.56 g (3.0 mmol) of 2-naphthaleneacetic acid (Nap), 1.14 g (3.0 mmol) of HBTU, and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, bubbled with nitrogen, and reacted at room temperature for 2 h. Filtered, and washed with DMF.

[0090] (6) 5 mL trifluoroacetic acid was added to the resin and reacted at room temperature for 2 h. The filtrate was obtained by filtration. The filtrate was added dropwise to 30 mL of ice-ethanol, and a white solid was obtained by filtration. The obtained crude product was purified by semi-preparative HPLC to obtain a polypeptide (NapKYpF) at a yield of 65%.

[0091] (7) 32 mg (0.0849 mmol) of TGN020-5ca, 9.67 mg (1.02 mmol) of N-hydroxysuccinimide, 13 mL (1.02

[0092] mmol) of N,N'-diisopropylcarbodiimide (DIC) were dissolved in 1 mL of DMF, and stirred at 37°C for 8 h.

[0093] (8) After the reaction was completed, 0.5 mL of water was added to quench the DIC. 58 mg (0.07 mmol) of NapKYpF and 23 mL

[0094] (0.14 mmol) of DIPEA were added, and stirred at 37°C for 2 h. The obtained mixture was purified by semi-preparative HPLC to obtain a final product (NapKYpF-TGN020) at a yield of 62%.

[0095] The structure of NapKYpF-TGN020 is as follows:

[0096]

[0097] Example 7: Preparation of NBD-AFFKYp-TGN020 of the polypeptide-TGN020 series material

[0098] (1) In a solid-phase synthesis tube, 1.0 g of 2-chloro-trityl resin was swelled in 10 mL of super-dry dichloromethane for 30 min. 1.3 g (3.0 mmol) of Fmoc-O-benzyl-L-phosphotyrosine (Yp) and 992 μL (6.0 mmol)

[0099] N,N-diisopropylethylamine (DIPEA) was dissolved in 5 mL of super-dry DMF and added to the resin, and reacted at room temperature for 12 h, filtered, and washed with DMF.

[0100] (2) 10 mL of methanol / dichloromethane (v / v=1:4) was added to the resin, and reacted at room temperature for 30 min, filtered, and washed with DMF. 10 mL of piperidine / DMF (v / v=1:4) was added to the resin, and reacted at room temperature for 30 min, filtered, and washed with DMF.

[0101] (3) 1.4 g (3.0 mmol) Fmoc-tert-butoxycarbonyl-L-lysine (K), 1.14 g (3.0 mmol) benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (HBTU) and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. 10 mL piperidine / DMF (v / v = 1 :4) was added to the resin and reacted for 30 min at room temperature. Filtration, DMF wash.

[0102] (4) 1.2 g (3.0 mmol) Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. 10 mL piperidine / DMF (v / v = 1 :4) was added to the resin and reacted for 30 min at room temperature. Filtration, DMF wash.

[0103] (5) 1.2 g (3.0 mmol) Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. 10 mL piperidine / DMF (v / v = 1 :4) was added to the resin and reacted for 30 min at room temperature. Filtration, DMF wash.

[0104] (6) 0.76 g (3.0 mmol) NBD-β-alanine (NBD-Ala), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin and reacted for 2 h at room temperature under nitrogen. Filtration, DMF wash.

[0105] (7) 5 mL trifluoroacetic acid was added to the resin and reacted for 2 h at room temperature. Filtration gave the filtrate. The filtrate was added dropwise to 30 mL ice-ethanol and filtration gave a white solid. The crude product was purified by semi-preparative HPLC to give the polypeptide (NBD-AFFKYp) in a yield of 50%.

[0106] (8) 32 mg (0.0849 mmol) TGN020-5ca, 9.67 mg (1.02 mmol) N-hydroxysuccinimide, 13 μL (1.02

[0107] (9) 0.5 mL DMF was added to the resin and reacted for 5 min at room temperature. Filtration, DMF wash. 0.5 mL 20% TFA / DMF was added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. The filtrate was added dropwise to 10 mL ice-ethanol and filtration gave a white solid. The crude product was purified by semi-preparative HPLC to give the polypeptide (TGN020-5ca) in a yield of 50%.

[0108] (10) 0.5 mL DMF was added to the resin and reacted for 5 min at room temperature. Filtration, DMF wash. 0.5 mL 20% TFA / DMF was added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. The filtrate was added dropwise to 10 mL ice-ethanol and filtration gave a white solid. The crude product was purified by semi-preparative HPLC to give the polypeptide (TGN020-5ca) in a yield of 50%.

[0109] (11) 0.5 mL DMF was added to the resin and reacted for 5 min at room temperature. Filtration, DMF wash. 0.5 mL 20% TFA / DMF was added to the resin and reacted for 2 h at room temperature. Filtration, DMF wash. The filtrate was added dropwise to 10 mL ice-ethanol and filtration gave a white solid. The crude product was purified by semi-preparative HPLC to give the polypeptide (TGN020-5ca) in a yield of 50%.

[0110] mmol) N,N'-diisopropylcarbodiimide (DIC) was dissolved in 1 mL of DMF and stirred at 37 °C for 8 h.

[0111] (9) After the reaction was completed, 0.5 mL of water was added to quench the DIC. 62.1 mg (0.07 mmol) of NBD-AFFKYp and 23 μL (0.14 mmol) of DIPEA were added to the system and stirred at 37 °C for 2 h. The resulting mixture was purified by semi-preparative HPLC to obtain the final product (NBD-AFFKYp-TGN020), yield: 59%.

[0112] The structure of NBD-AFFKYp-TGN020 is shown below:

[0113]

[0114] Example 8 Preparation of NBD-AFFYpKK-TGN020 of the polypeptide-TGN020 series material

[0115] (1) In a solid-phase synthesis tube, 1.0 g of 2-chloro-trityl resin was swelled in 10 mL of super dry dichloromethane for 30 min. 1.4 g (3.0 mmol) of Fmoc-tert-butoxycarbonyl-L-lysine (K) and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, and the reaction was carried out at room temperature for 12 h. Filtration and DMF washing were performed.

[0116] N,N-diisopropylethylamine (DIPEA) was dissolved in 5 mL of super dry DMF and added to the resin, and the reaction was carried out at room temperature for 12 h. Filtration and DMF washing were performed.

[0117] (2) 10 mL of methanol / dichloromethane (v / v = 1:4) was added to the resin, and the reaction was carried out at room temperature for 30 min. Filtration and DMF washing were performed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, and the reaction was carried out at room temperature for 30 min. Filtration and DMF washing were performed.

[0118] (3) 1.4 g (3.0 mmol) of Fmoc-tert-butoxycarbonyl-L-lysine (K), 1.14 g (3.0 mmol) of benzotriazole

[0119] -N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 992 μL (6.0 mmol) of DIPEA were dissolved in 5 mL of DMF and added to the resin, and the reaction was carried out at room temperature for 2 h. Filtration and DMF washing were performed. 10 mL of piperidine / DMF (v / v = 1:4) was added to the resin, and the reaction was carried out at room temperature for 30 min. Filtration and DMF washing were performed.

[0120] (4) 1.3 g (3.0 mmol) Fmoc-O-benzyl-L-phosphotyrosine (Yp), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing.

[0121] (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing.

[0122] 10 mL piperidine / DMF (v / v = 1 :4) were added to the resin, and reacted at room temperature for 30 min. Filtration, DMF washing.

[0123] (5) 1.2 g (3.0 mmol) Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing.

[0124] (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing. 10 mL piperidine / DMF (v / v = 1 :4) were added to the resin, and reacted at room temperature for 30 min. Filtration, DMF washing.

[0125] (6) 1.2 g (3.0 mmol) Fmoc-L-phenylalanine (F), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing.

[0126] (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h. Filtration, DMF washing. 10 mL piperidine / DMF (v / v = 1 :4) were added to the resin, and reacted at room temperature for 30 min. Filtration, DMF washing.

[0127] (7) 0.76 g (3.0 mmol) NBD-β-alanine (NBD-Ala), 1.14 g (3.0 mmol) HBTU and 992 μL (6.0 mmol) DIPEA were dissolved in 5 mL DMF and added to the resin, and reacted at room temperature for 2 h under nitrogen. Filtration, DMF washing.

[0128] (8) 5 mL trifluoroacetic acid were added to the resin, and reacted at room temperature for 2 h. Filtration gave a filtrate. The filtrate was added dropwise to 30 mL ice-ethanol, and filtration gave a white solid. The obtained crude product was purified by semi-preparative HPLC to give a polypeptide (NBD-AFFYpKK), yield: 40%.

[0129] (9) 32 mg (0.0849 mmol) TGN020-5ca, 9.67 mg (1.02 mmol) N-hydroxysuccinimide, 13 μL (1.02

[0130] 1 mmol) N,N′-diisopropylcarbodiimide (DIC) was dissolved in 1 mL LDM and heated and stirred at 37 °C for 8 h.

[0131] (10) After the reaction was complete, 0.5 mL of water was added to quench DIC. 30 mg (0.035 mmol) of NBD-AFFYpKK and 23 μL (0.14 mmol) of DIPEA were added to the system, and the mixture was heated and stirred at 37 °C for 2 h. The resulting mixture was purified by semi-preparative HPLC to obtain the final product (NBD-AFFYpKK-TGN020), yield: 40%.

[0132] The structure of NBD-AFFYpKK-TGN020 is as follows:

[0133]

[0134] Test Example 1: Nuclear Magnetic Resonance and Electrospray Mass Spectrometry (ESI-MS) Tests

[0135] The polypeptide-TGN020 (NapFFKYp-OMe-TGN020) prepared in Example 4 was subjected to... 1 H-NMR and ESI-MS tests yielded Figure 1 and Figure 2 .

[0136] Figure 1 For NapFFKYp-OMe-TGN020 1 H-NMR spectrum. Figure 1 The characteristic hydrogen δ9.25 (s, 1H) of TGN020 and the α hydrogen δ4.61–4.40 (m, 4H) on the polypeptide backbone show that the polypeptide and TGN020 were covalently coupled in a 1:1 ratio, proving that NapFFKYp-OMe-TGN020 was successfully obtained.

[0137] Figure 2 The image shows the electrospray ionization-mass spectra (ESI-MS) of the NapFFKYp-OMe-TGN020. From... Figure 2 As can be seen from the mass spectrometry data, this molecule [M+H] + ] + ,1225.30,[MH + ] - The result of obtaining 1223.30 proves that the molecular weight of the molecule we finally obtained is 1224.30, which is consistent with the molecular weight calculated by Chembiodraw software, proving that we have successfully obtained NapFFKYp-OMe-TGN020.

[0138] Test Example 2: Cumulative Performance Test of Peptide-TGN020 Outside the Cell Membrane

[0139] (1) Test purpose

[0140] The morphology of the cell membrane of glioma cells U87MG and normal cells HUVEC was imaged by scanning electron microscopy and laser confocal microscopy, and the assembly morphology and accumulation performance of the polypeptide-TGN020 (NapFFKYp-OMe-TGN020) prepared in Example 4 on the surface of the glioma cell membrane were observed.

[0141] (2) Test method

[0142] 2x10 5 U87MG cells were plated in a 20 mm confocal dish overnight. The culture medium was discarded, and 1 mL of DMEM medium containing 200 μM NapFFKYp-OMe-TGN020 was added for 24 h. The cells were washed with PBS, fixed with 4% paraformaldehyde solution, gradient dehydrated with water-ethanol solution, fixed on the sample stage, gold sprayed, and the cell membrane surface was imaged using a super high resolution cold field emission scanning electron microscope SU8220. The resulting photos were processed using ImageJ. 2x10 5 U87MG or HUVEC cells were plated in a 20 mm confocal dish overnight. The culture medium was discarded, and 1 mL of DMEM medium containing 200 μM NBD-AFFKYp-OMe-TGN020 was added for 2 h. The cells were washed with PBS, and serum-free DMEM medium was added. The cells were imaged using a laser confocal microscope, and the resulting photos were processed using ImageJ.

[0143] (3) Test results

[0144] Figure 3 The polypeptide-TGN020 morphology on the surface of the glioma cell U87MG cell membrane was photographed by scanning electron microscopy. The Control group was without polypeptide-TGN020 (NapFFKYp-OMe-TGN020) as a negative control. As can be seen from Figure 3 , compared with no polypeptide-TGN020, after adding polypeptide-TGN020, nanofiber assembly was enriched on the surface of the glioma cell membrane.

[0145] Figure 4Figure 1 shows the distribution of polypeptide-TGN020 (NBD-AFFKYp-OMe-TGN020) on the cell membrane of glioma cell U87MG taken by laser confocal microscope. NBD represents the green fluorescence channel of NBD, Bright represents the bright field channel, and Merge is the superposition of the fluorescence channel and the bright field channel. NBD-AFFKYp-OMe-TGN020 is a polypeptide molecule with TGN020 ligand, and NBD-AFFKYp-OMe is a polypeptide molecule without TGN020 ligand for control. It can be seen from Figure 4 that the polypeptide alone cannot be enriched on the cell membrane surface of glioma cell U87MG and cannot achieve the targeting function of U87MG cell membrane. The polypeptide molecule with TGN020 ligand can be enriched on the cell membrane surface of U87MG and can achieve the targeting function of U87MG cell membrane. Neither the polypeptide alone nor the polypeptide molecule with TGN020 ligand can be enriched on the cell membrane surface of HUVEC. It is shown that the polypeptide molecule with TGN020 ligand can target the glioma cell membrane without affecting normal cells.

[0146] In summary, the polypeptide-TGN020 of the present application can be targeted to the surface of glioma cell membrane for enrichment, and has the technical effect of realizing the targeting and accumulation of polypeptide assembly on the glioma cell membrane.

[0147] Test Example 3: Long-acting inhibition test of aquaporin 4 (AQP4) on glioma cells

[0148] (1) Purpose of the test

[0149] The rate of water in and out of glioma cell U87MG was quantified and characterized by magnetic resonance imaging technology, and the inhibition effect of the final product polypeptide-TGN020 (NapFFKYp-OMe-TGN020) in Example 4 on the water permeation rate of aquaporin 4 (AQP4) on the surface of glioma cell membrane was determined.

[0150] (2) Test method

[0151] 1×10 5U87MG glioma cells were cultured in 0.1 mL DMEM medium containing 14 μM polypeptide-TGN020 (NapFFKYp-OMe-TGN020) for 72 h, dissociated by adding 1 ml 0.25% trypsin-EDTA solution to a 60 mm culture dish and incubating for 0.5-1.0 min, and then resuspended with 2 mL PBS (or PBS supplemented with 5 mM Gd). After centrifuging the cell sample at 150 g for 5 min at 4 °C, the sample was resuspended in a custom MR-compatible glass sample tube (5 mm in diameter x 20 mm in length) containing about 250-300 μL PBS (or PBS + 5 mM Gd). We then centrifuged the MRI tube at 300 g for 2 min at 4 °C before performing MRI. The rate of water molecules entering and exiting AQP4 on the U87MG cell membrane was detected using the magnetic resonance imaging method.

[0152] (3) Test results

[0153] Figure 5 Quantification of the rate of water molecules entering and exiting the cell for magnetic resonance imaging io The quantification graph. The Control group is the negative control group without any material, the NapFFKYp-OMe-TGN020 group is the polypeptide molecule with the TGN020 ligand added, the NapFFKYp-OMe group is the polypeptide molecule alone without the TGN020 ligand, and the TGN020 group is the small molecule inhibitor alone without the polypeptide molecule.

[0154] From Figure 5 It can be seen that the polypeptide molecule NapFFKYp-OMe-TGN020 with the TGN020 ligand can reduce the water permeation rate of AQP4 protein at a long-acting time of 72 h compared with the control group, while the polypeptide molecule NapFFKYp-OMe without the TGN020 ligand and the small molecule inhibitor TGN020 alone without the polypeptide molecule cannot reduce the water permeation rate of AQP4 protein at a long-acting time of 72 h.

[0155] Therefore, the polypeptide-TGN020 (NapFFKYp-OMe-TGN020) of the present application can achieve long-acting inhibition of AQP4 on U87MG cells, while the polypeptide molecule alone and the small molecule TGN020 alone cannot achieve long-acting inhibition of AQP4 on U87MG cells.

[0156] Test example 4: cytotoxicity test on glioma cells U87MG

[0157] (1) Purpose of the test

[0158] The cytotoxicity and growth inhibition of the final product polypeptide-TGN020 (NapFFKYp-OMe-TGN 020) in Example 4 on glioma cells were determined by MTT cytotoxicity experiment.

[0159] (2) Test method

[0160] The final product polypeptide-TGN020 (NapFFKYp-OMe-TGN020) in Example 4, TGN020 and the final product polypeptide (NapFFKYp-OMe) in Example 3 were prepared into 100 μM solution in DMEM complete medium, and were diluted by ten times, 200 μL was added to each well in a 96-well plate. The digested U87MG cells were prepared into a cell suspension with a cell density of 3 x 10 6 , and 10 μL was added to each well. After the 96-well plate was placed in a cell incubator for 72 hours, the culture medium was aspirated, 100 μL of MTT culture solution was added, and after incubation for 4 hours, the MTT solution was aspirated, 110 μL of Formazan dissolving solution was added, and after thorough dissolution, the absorbance at 470 nm was determined using an enzyme marker, and the absorbance value of each well was plotted using Graphpad to obtain the killing effect curve.

[0161] (3) Test results

[0162] Figure 6 The killing effect curve of the final product polypeptide-TGN020 (NapFFKYp-OMe-TGN020) in Example 4, the small molecule inhibitor TGN020 and the final product polypeptide (NapFFKYp-OMe) in Example 3 on glioma cells U87MG at a concentration gradient.

[0163] It can be seen from Figure 6 that:

[0164] The half-inhibitory concentration of the polypeptide molecule NapFFKYp-OMe alone without the TGN020 ligand (circular line) was 99.53 μM.

[0165] The small molecule inhibitor TGN020 alone without the polypeptide molecule (square line) did not detect cytotoxicity within 100 μM.

[0166] The half-inhibitory concentration of the polypeptide molecule NapFFKYp-OMe-TGN020 with the TGN020 ligand (triangular line) was 14.18 μM.

[0167] In summary, the killing effect of the NapFFKYp-OMe-TGN020 of the present application on glioma cells U87MG is better than that of the polypeptide alone and that of TGN020 alone. It is illustrated that the polypeptide-TGN020 obtained by coupling TGN020 by the method of the present application has the ability of target enrichment and long-term inhibition of AQP4, and further has more excellent tumor killing ability.

[0168] Test Example 5: Test of temozolomide chemotherapy sensitization on ectopic glioma

[0169] (1) Test purpose

[0170] The final product polypeptide-TGN020 (NapFFKYp-OMe-TGN020) in Example 4 was verified by a nude mouse subcutaneous tumor experiment to have the ability to inhibit the proliferation of glioma and the sensitization effect of the chemotherapy drug temozolomide.

[0171] (2) Test method

[0172] Nude mouse subcutaneous tumor experiment. BALB / c nude mice were subcutaneously inoculated with 5x10 6 U87MG cells, and one week later, when the subcutaneous tumor volume reached 50mm 3 , 1mM temozolomide 500μL was intraperitoneally injected in a 5-day administration and 2-day rest manner, and 1mM NapFFKYp-OMe-TGN020 and TGN020 alone 100μL were intratumorally injected in a 2-day / once administration manner. The change in tumor volume of the subcutaneous tumor of the mice was recorded every day for 14 days.

[0173] (3) Test results

[0174] Figure 7 The nude mouse subcutaneous tumor volume change curve under the combined administration of polypeptide-TGN020 (NapFFKYp-OMe-TGN020), small molecule inhibitor TGN020 and temozolomide. The untreated group is PBS+PBS (circular line); the polypeptide alone group is PBS+NapFFKYp-OMe-TGN020 (equilateral triangle line); the temozolomide alone group is TMZ+PBS (square line); the group of temozolomide combined with small molecule inhibitor is TMZ+TGN020 (inverted triangle line); the group of temozolomide combined with polypeptide-TGN020 is TMZ+NapFFKYp-OMe-TGN020 (hexagon line).

[0175] From Figure 7 it can be seen that:

[0176] Under the treatment of NapFFKYp-OMe-TGN020 alone (equilateral triangle line), there is no significant difference in tumor volume compared with the untreated group, and it does not have the ability to directly inhibit the proliferation of glioma.

[0177] Under the treatment of temozolomide alone (square line), the tumor volume change has partial inhibition compared with the untreated group, but the tumor volume still grows slowly.

[0178] Under the treatment of temozolomide and small molecule inhibitor TGN020 (inverted triangle line) combination, the tumor volume change has no significant difference compared with the temozolomide alone group, and the tumor volume still grows slowly, and the small molecule inhibitor TGN020 does not have the ability of directly inhibiting glioma proliferation and sensitizing temozolomide.

[0179] Under the treatment of NapFFKYp-OMe-TGN020 and temozolomide combination (hexagon line), the tumor volume is obviously inhibited compared with the untreated group and the temozolomide alone treatment, and therefore, the NapFFKYp-OMe-TGN020 can sensitize temozolomide chemotherapy drugs and has the ability of inhibiting glioma proliferation.

[0180] Therefore, the polypeptide-TGN020 can effectively perform chemotherapy sensitization on the chemotherapy drug temozolomide, so as to synergistically kill and inhibit glioma.

[0181] Finally, it should be explained that the above preferred embodiments are only used to illustrate the technical solutions of the present application but not limit the present application, and although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for preparing a targeted drug peptide for glioma treatment, characterized in that, The preparation steps are as follows: 1) Preparation of a small molecule inhibitor of AQP4, TGN020-5ca, wherein the structural formula of TGN020-5ca is as follows: 2) Preparation of polypeptides or methylated polypeptides: Different amino acids are added sequentially in a solid-phase synthesis tube to obtain polypeptides. The polypeptides are then reacted with trimethylbromosilane and methanol to obtain methylated polypeptides. 3) Couple a small molecule inhibitor of AQP4 to a peptide molecule to prepare peptide-TGN020: First, add N-hydroxysuccinimide and N,N′-diisopropylcarbodiimide to activate the carboxyl group on TGN020-5ca at 37℃~60℃ for 6~8 hours. Then, add the peptide or methylated peptide and N,N-diisopropylethylamine and react at 25℃~37℃ for 1~2 hours. The molar ratio of TGN020-5ca to the peptide or methylated peptide is 1.2:1~1.5:

1.

2. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 1, characterized in that, In step 3), based on the amount of the polypeptide or methyl esterified polypeptide, the amount of N-hydroxysuccinimide added is 1.5 equivalents, the amount of N,N′-diisopropylcarbodiimide added is 3 equivalents, and the amount of N,N-diisopropylethylamine added is 3 equivalents.

3. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 1, characterized in that, The sequence of the polypeptide in step 2) is NapFFKYp or NapKYpF or NBD-AFFKYp or NBD-AFFYpKK.

4. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 1, characterized in that, Step 1) specifically involves: 1) Preparation of ethyl 5-[3-(tert-butoxycarbonylamino)prop-1-yn-1-yl]pyridine-2-carboxylic acid; 2) Prepare ethyl 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylate using the nicotinic acid from step 1). 3) Prepare 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid using ethyl 5-[3-(isopropoxycarbonylamino)propyl]pyridine-2-carboxylic acid from step 2); 4) Dissolve the 5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxylic acid from step 3) in acetonitrile, add 2-amino-1,3,4-thiadiazole, N,N,N′,N′-tetramethylchloromethacin hexafluorophosphate and N-methylimidazolium, stir the mixture at room temperature for 8-12 h, rotary evaporate the resulting mixture to obtain a solid, extract with ethyl acetate and saturated brine, adjust the pH to 2-4 with HCl, collect the organic phase and rotary evaporate, purify the solid residue by silica gel column chromatography to obtain N-(2-amino-1,3-thiazol-5-yl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide; 4) The N-(2-amino-1,3-thiazolyl)-5-[3-(tert-butoxycarbonylamino)propyl]pyridine-2-carboxamide from step 4) was dissolved in water, trifluoroacetic acid and methanol were added, and the mixture was stirred at room temperature for 6-8 h. The solvent was removed by rotary evaporation to obtain a white solid, which was dissolved in DMF, and glutaric anhydride and N,N-diisopropylethylamine were added. The mixture was stirred at room temperature for 6-8 h, eluted and purified, and freeze-dried to obtain TGN020-5ca.

5. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 3, characterized in that, When the polypeptide sequence in step 2) is NapFFKYp, the preparation method is as follows: 2-chloro-triphenylmethyl chloride resin is swollen in ultra-dry dichloromethane. Fmoc-O-benzyl-L-phosphotyrosine and N,N-diisopropylethylamine are dissolved in DMF and added to the resin. The reaction is carried out at room temperature for 6-12 h, filtered, washed, and then methanol / dichloromethane (v / v = 1:4) is added. The reaction is carried out at room temperature for 30 min, filtered, washed with DMF, and protected with 20% piperidine to remove Fmoc. Then, L-lysine, L-phenylalanine, L-phenylalanine, and 2-naphthaleneacetic acid are added sequentially in the ratio of resin:amino acid:HBTU:DIPEA = 1:3:3:

6. Washing and 20% piperidine are used to remove Fmoc at each step. Finally, the resin is cleaved with trifluoroacetic acid and precipitated with ice-cold diethyl ether. The mixture is purified by HPLC and lyophilized to obtain the polypeptide molecule.

6. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 4, characterized in that, When the polypeptide sequence in step 2) is NapKYpF, the preparation method is as follows: 2-chloro-triphenylmethyl chloride resin is swollen in ultra-dry dichloromethane. Fmoc-L-phenylalanine and N,N-diisopropylethylamine are dissolved in DMF and added to the resin. The reaction is carried out at room temperature for 6-12 h. After filtration and washing, methanol / dichloromethane (v / v = 1:4) is added and the reaction is carried out at room temperature for 30 min. After filtration and washing with DMF, Fmoc is deprotected with 20% piperidine. Then, Fmoc-O-benzyl-L-phosphotyrosine, L-lysine, and 2-naphthaleneacetic acid are added sequentially in the ratio of resin:amino acid:HBTU:DIPEA = 1:3:3:

6. Washing and deprotection with 20% piperidine are performed at each step. Finally, the resin is cleaved with trifluoroacetic acid and precipitated with ice-cold diethyl ether. After HPLC purification and lyophilization, the polypeptide molecule is obtained.

7. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 4, characterized in that, When the polypeptide sequence in step 2) is NBD-AFFKYp, the preparation method is as follows: 2-chloro-triphenylmethyl chloride resin is swollen in ultra-dry dichloromethane. Fmoc-O-benzyl-L-phosphotyrosine and N,N-diisopropylethylamine are dissolved in DMF and added to the resin. The reaction is carried out at room temperature for 6-12 h, filtered, washed, and then methanol / dichloromethane is added. The reaction is carried out at room temperature for 30 min, filtered, washed with DMF, and protected with 20% piperidine to remove Fmoc. Then, L-lysine, L-phenylalanine, L-phenylalanine, and NBD-β-alanine are added sequentially in the ratio of resin:amino acid:HBTU:DIPEA = 1:3:3:

6. Washing and 20% piperidine are used to remove Fmoc at each step. Finally, the resin is cleaved with trifluoroacetic acid and precipitated with ice-cold diethyl ether. The mixture is purified by HPLC and lyophilized to obtain the polypeptide molecule.

8. The method for preparing a targeted drug polypeptide for glioma treatment according to claim 4, characterized in that, When the polypeptide sequence in step 2) is NBD-AFFYpKK, the preparation method is as follows: 2-chloro-triphenylmethyl chloride resin is swollen in ultra-dry dichloromethane. Fmoc-L-lysine and N,N-diisopropylethylamine are dissolved in DMF and added to the resin. The reaction is carried out at room temperature for 6-12 h, filtered, washed, and then methanol / dichloromethane is added. The reaction is carried out at room temperature for 30 min, filtered, washed with DMF, and protected with 20% piperidine to remove Fmoc. Then, L-lysine, Fmoc-O-benzyl-L-phosphotyrosine, L-phenylalanine, L-phenylalanine, and NBD-β-alanine are added sequentially in a resin:amino acid:HBTU:DIPEA ratio of 1:3:3:

6. Each step involves washing and protecting with 20% piperidine to remove Fmoc. Finally, the resin is cleaved with trifluoroacetic acid and precipitated with ice-cold diethyl ether. The mixture is purified by HPLC and lyophilized to obtain the polypeptide molecule.

9. The drug polypeptide prepared by the method for preparing a targeted drug polypeptide for glioma treatment according to any one of claims 1-8.

10. The application of the targeted drug polypeptide for glioma treatment according to claim 9 as a sensitizer for chemotherapeutic drugs that inhibit glioma cells, characterized in that, Used to inhibit aquaporin 4, with an inhibition time of ≥72 hours.

Images