A supramolecular assembly with spermine response and its preparation method and application

Abstract

The present invention discloses a supramolecular assembly with spermine response, a preparation method and application thereof, and belongs to the technical field of material preparation. The present invention obtains supramolecular assemblies (SNPs) by self-assembling a bipyridinium salt diphenylanthracene derivative (PyEn) and a water-soluble pillar[5]arene (WP5C5) in water. The anthracycline derivative with high cytotoxicity as the assembly unit in the supramolecular assembly is used as an anti-tumor agent, and the longer alkyl chain in the water-soluble pillar[5]arene shields the toxicity of the anthracycline derivative, thereby reducing the toxicity to normal cells. The spermine in spermine-overexpressing cancer cells is used to competitively release the anthracycline derivative in the supramolecular assembly, thereby restoring the toxicity to cancer cells. The supramolecular assembly prepared by the present invention has high negative charge and good stability, and also has the advantages of good biocompatibility and responsiveness to the tumor marker spermine stimulation.

Description

Technical Field

[0001] The present invention belongs to the technical field of material preparation, and in particular relates to a supramolecular assembly with spermine response, a preparation method and an application thereof. Background Art

[0002] Among the many cancer treatments, traditional chemotherapy remains one of the most effective. Chemotherapy drugs are often combined with drug delivery vehicles to enhance therapeutic efficacy while minimizing drug toxicity. Common drug delivery vehicles include inorganic and organic nanocarriers. However, inorganic nanocarriers often have poor biocompatibility, are difficult to degrade, and have potential long-term biotoxicity. Organic nanocarriers, on the other hand, are less toxic and can be coupled to multiple drugs. However, this stable loading results in slow drug release and insufficient drug accumulation at the tumor site, resulting in limited therapeutic efficacy.

[0003] In recent years, supramolecular chemotherapy has successfully combined supramolecular chemistry with traditional chemotherapy, opening up new avenues for the delivery of chemotherapeutic drugs. Supramolecular chemotherapy systems responsive to the tumor microenvironment have been widely constructed, such as those responsive to glutathione (GSH), hydrogen peroxide (H2O2), adenosine triphosphate (ATP), esterases, and tumor markers. Currently, there are no supramolecular chemotherapy systems specifically responsive to the tumor marker SPM. Therefore, it is of great significance to explore intelligent nanocarriers with excellent biocompatibility for drug delivery and precise controlled release to achieve excellent therapeutic effects while minimizing toxic side effects to normal tissues. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a supramolecular assembly with spermine response and its preparation method and application, so as to solve the technical problem that the targeting accuracy of existing drugs for treating tumors is not high enough.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide a method for preparing a supramolecular assembly with spermine response, wherein a bipyridinium diphenylanthracene derivative and a water-soluble pillar[5]arene are self-assembled in water to obtain a supramolecular assembly.

[0006] The present invention utilizes the uniquely sized hydrophobic cavity of the supramolecular macrocycle to encapsulate chemotherapy drugs that match its cavity. This significantly increases the water solubility and stability of the chemotherapy drugs through host-guest interactions. Furthermore, the macrocyclic molecules can further combine with functional targeting objects to form drug-loaded assemblies with active tumor targeting. Through passive targeting via the EPR effect and active targeting of the targeting unit, efficient drug enrichment in tumor tissue can be achieved, further enhancing the therapeutic effect. The dynamic nature of non-covalent interactions endows supramolecular chemotherapy with excellent stimulus-responsive properties. In particular, responding to the tumor microenvironment can promote the precise in situ release of drugs in tumor tissue, thereby achieving better therapeutic effects while reducing toxic side effects on normal tissues.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows:

[0008] Furthermore, the preparation method comprises the following steps:

[0009] S1. preparing a bipyridinium salt diphenylanthracene derivative;

[0010] S101, mixing a diphenylanthracene compound, a boric acid compound, a palladium catalyst, and a base in an organic solvent at a molar ratio of 12-17:12-17:2-4:80-120, and reacting under a protective atmosphere at 110-120° C. for 8-12 hours to obtain product 1;

[0011] The diphenylanthracene compound serves as a halogenated aromatic hydrocarbon substrate in the reaction and contains a halogen atom that can undergo a coupling reaction. This halogen atom will be replaced during the reaction. The boronic acid compound serves as a coupling reagent to provide a benzene ring portion in the reaction. Under alkaline conditions, the boronic acid compound undergoes a coupling reaction with the halogenated aromatic hydrocarbon substrate, activating the organic boron intermediate to form a new carbon-carbon bond, which is usually manifested as the formation of an organic borate or a corresponding tetracoordinate boron compound. The palladium catalyst (usually a zero-valent palladium catalyst, such as Pd(0)) reacts with the halogenated aromatic hydrocarbon, and the palladium enters the carbon-halogen bond of the substrate to form palladium-carbon and palladium-halide intermediates. Migration insertion: The organic group of the boronic acid compound is transferred to the palladium to form a new palladium-carbon bond. The intermediate palladium compound is connected by two organic groups. Reductive elimination: The palladium catalyst generates a newly formed carbon-carbon bond between the two organic groups and recovers it in the form of palladium(0). The palladium catalyst is recycled. Finally, the product 1 with a large aromatic structure is obtained.

[0012] S102, dissolving the product 1 in an organic solvent, and then adding a demethylation reagent to react for 8-12 hours to obtain the product 2; the ratio of the product 1 to the demethylation reagent is 3.5-4 mmol: 0.8-1.2 mL;

[0013] S103, product 2, a bromine-containing compound, and a base are mixed in an organic solvent at a molar ratio of 1-2:10-20:10-20, and then reacted under a protective atmosphere at 70-90° C. for 10-14 hours to obtain product 3;

[0014] Product 2 acts as a nucleophile to attack the carbon atom of the electrophile (bromine-containing compound) from the back, causing a nucleophilic substitution reaction of the halogenated hydrocarbon, and ultimately obtaining product 3 with long alkyl chains at both ends of the anthracene ring.

[0015] S104, mixing the product tri with pyridine in a ratio of 0.3-0.5 mmol: 3-7 mL, and reacting at 70-90° C. for 7-9 hours to obtain a crude product; mixing the crude product with an ammonium hexafluorophosphate solution to react for 20-40 minutes, filtering, drying, and mixing the dried product with a tetrabutylammonium chloride solution to react for 20-40 minutes to obtain a bipyridinium diphenylanthracene derivative (PyEn); the molar ratio of the crude product, the ammonium hexafluorophosphate solution, and the tetrabutylammonium chloride solution is 1:2-5:2-5;

[0016] The hexafluorophosphate ions in the aqueous ammonium hexafluorophosphate solution can be exchanged with the counter ions (halogen atoms) in the crude product, and the chloride ions in the tetrabutylammonium chloride solution can be exchanged with the hexafluorophosphate of the product to obtain a bipyridinium diphenylanthracene derivative.

[0017] S2, preparation of water-soluble column [5] aromatic hydrocarbons (WP5C5);

[0018] S3. A bipyridinium diphenylanthracene derivative (PyEn) and a water-soluble pillar[5]arene (WP5C5) were mixed at a molar ratio of 1:0.5-6 and reacted for 10-14 hours to obtain supramolecular assemblies (SNPs).

[0019] Furthermore, the diphenylanthracene compound is 9,10-dibromoanthracene; the boronic acid compound is 4-methoxy-2-methylphenylboronic acid; the palladium catalyst is tetrakistriphenylphosphine palladium or dichlorobis(triphenylphosphine)palladium(II); and the base is sodium carbonate, potassium carbonate or potassium hydroxide.

[0020] Furthermore, the protective atmosphere is composed of argon or nitrogen.

[0021] Furthermore, the demethylation reagent is boron tribromide.

[0022] Furthermore, the bromine-containing compound is 1,6-dibromohexane, ethyl 5-bromovalerate or ethyl bromoacetate.

[0023] Further, step S2 specifically includes:

[0024] S201, mixing hydroquinone and a base in an organic solvent, then adding a bromine-containing compound, and reacting at 90-110° C. for 46-50 hours to obtain product 4; the molar ratio of hydroquinone, base and bromine-containing compound is 3-5:22-26:8-10;

[0025] Hydroquinone acts as a nucleophile, and the bromine-containing compound acts as an electrophile, resulting in a nucleophilic substitution reaction with the halogenated hydrocarbon. The nucleophile attacks the carbon atom from the back, causing the bromine atom to leave, ultimately yielding product 4 with long alkyl chains at both ends of the benzene ring.

[0026] S202, mixing the product 4 and paraformaldehyde in an organic solvent, then adding boron trifluoride etherate and polymerizing for 25-35 minutes to obtain the product 5; the ratio of the product 4, paraformaldehyde and boron trifluoride etherate is 1-3 mmol: 5-7 mmol: 1-3 mmol;

[0027] Paraformaldehyde typically undergoes a condensation reaction with phenol or its derivatives to form a methyl-bridged aromatic ring structure. Boron trifluoride ether forms an adduct with formaldehyde, increasing the electrophilicity of formaldehyde and making it more susceptible to reaction with phenol. It also promotes the activation of phenol and increases its reactivity by interacting with the hydroxyl groups in phenol. Under the catalysis of boron trifluoride ether, formaldehyde and phenol undergo a condensation reaction to form a methyl-bridged phenol unit, ultimately yielding columnar[5] aromatic hydrocarbons containing long alkyl chains.

[0028] S203, dissolving the product five and the base in an organic solvent at a molar ratio of 0.5-1.5:25-35, hydrolyzing at 50-70° C. for 10-14 hours, and then adjusting the pH value of the reaction system to 1.5-2.5 to obtain the product six;

[0029] S204. The product hexahydrate and the base are mixed at a molar ratio of 0.5-1.5:8-12 and reacted for 0.5-1.5 hours to obtain water-soluble columnar [5] aromatic hydrocarbons (WP5C5).

[0030] Furthermore, in step S3, the molar ratio of the bipyridinium salt diphenylanthracene derivative and the water-soluble pillar[5]arene is 1:1.

[0031] The invention also discloses a spermine-responsive supramolecular assembly prepared by the preparation method.

[0032] The invention also discloses the application of the supramolecular assembly with spermine response in the preparation of drugs for treating tumors.

[0033] Furthermore, the tumor treatment drug is an injectable drug.

[0034] Furthermore, the injection method is intratumoral injection or tail vein injection.

[0035] The beneficial effects of the present invention are as follows: the present invention utilizes anthracycline derivatives with high cytotoxicity as an assembly unit in a supramolecular assembly as an anti-tumor agent, utilizes the longer alkyl chain in the water-soluble columnar [5] aromatic hydrocarbon of the assembly unit in the supramolecular assembly to shield the toxicity of the anthracycline derivatives, and reduces the toxicity to normal cells; utilizes spermine in cancer cells overexpressing spermine to competitively release the anthracycline derivatives in the supramolecular assembly, and restores the toxicity to cancer cells. The obtained supramolecular assembly has high negative charge and good stability, and also has the advantages of good biocompatibility and responsiveness to the tumor marker spermine stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the particle size distribution diagram of supramolecular assembly SNPs;

[0037] Figure 2 Transmission electron microscopy image of supramolecular assembly SNPs;

[0038] Figure 3 To evaluate the stability of supramolecular assembly SNPs in water;

[0039] Figure 4 The H NMR spectra of supramolecular assembly SNPs under different spermine equivalents;

[0040] Figure 5 The fluorescence spectra of supramolecular assembly SNPs under different spermine equivalents;

[0041] Figure 6 To evaluate the uptake behavior of supramolecular assembly SNPs into cancer cells;

[0042] Figure 7 To evaluate the subcellular organelle targeting of supramolecular assembly SNPs;

[0043] Figure 8 To evaluate the cytotoxicity of supramolecular assembly SNPs to HK2 normal cells;

[0044] Figure 9 To evaluate the cytotoxicity of supramolecular assembly SNPs to A549 cancer cells;

[0045] Figure 10 To evaluate the toxicity of supramolecular assembly SNPs to HCT116 cancer cells;

[0046] Figure 11 Figure 2 is a graph showing the changes in tumor weight after tail vein injection of supramolecular assembly SNPs and the control group;

[0047] Figure 12 Figure 2 shows the weight changes of mice after tail vein injection of supramolecular assembly SNPs and the control group;

[0048] Figure 13 H&E staining images of sections of major organs of mice after treatment with supramolecular assembly SNPs. DETAILED DESCRIPTION

[0049] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0050] Example 1

[0051] A method for preparing a supramolecular assembly responsive to spermine comprises the following steps:

[0052] S1. Preparation of a bipyridinium salt diphenylanthracene derivative (PyEn), the reaction equation is as follows:

[0053]

[0054] S101. 9,10-dibromoanthracene (1.00 g, 2.99 mmol), 4-methoxy-2-methylphenylboronic acid (1.00 g, 2.99 mmol), tetrakistriphenylphosphine palladium (0.702 g, 0.608 mmol) and sodium carbonate (10 mL, 2 mol / L) were mixed in 40 mL of an organic solvent (the volume ratio of ethanol to toluene was 1:3), and the mixture was stirred at 115°C under an argon atmosphere for 10 h. After the reaction solution was cooled to room temperature, it was filtered, the filtrate was collected, and the solvent was removed by distillation under reduced pressure. The crude product solid was dissolved in dichloromethane (80 mL), extracted with water, and the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered again. The solvent was removed by distillation under reduced pressure, and the product was purified by flash column chromatography on a silica gel column (the volume ratio of dichloromethane to petroleum ether was 1:1) to obtain product 1 as a white powder (887 mg, yield 70%).

[0055] S102, product 1 (780 mg, 1.86 mmol) was dissolved in anhydrous dichloromethane (50 mL), and boron tribromide (0.5 mL) was added dropwise to the reaction system under argon protection and ice bath conditions to carry out demethylation reaction. After 10 h of reaction, deionized water (20 mL) was added to quench the reaction, and a light yellow solid precipitated. The solid was filtered and washed with methanol, and the filter cake was decompressed to remove the solvent to obtain product 2 as a light yellow powder (587 mg, yield 81%);

[0056] S103, product 2 (300 mg, 0.769 mmol), 1,6-dibromohexane (1.86 g, 7.69 mmol), anhydrous potassium carbonate (1.06 g, 7.69 mmol) and acetone (30 mL) were mixed uniformly, and then a nucleophilic substitution reaction was carried out at 80° C. under an argon atmosphere for 12 h. After the reaction solution was cooled to room temperature, potassium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to remove the solvent. The obtained solid was dissolved in dichloromethane, and after extraction and separation with water, the organic phase was washed with saturated brine (30 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered again, and the solvent was distilled under reduced pressure. After passing through a silica gel column for flash column chromatography (the chromatographic solvent was petroleum ether), a white powder of product 3 was obtained;

[0057] S104. Product 3 (300 mg, 0.420 mmol) was mixed with pyridine (5 mL), and the mixture was reacted at 80° C. for 8 h. After the reaction solution was cooled to room temperature, the solvent was distilled off under reduced pressure, and the obtained solid crude product was washed with ethyl acetate to obtain a bromide salt of the product;

[0058] The bromide salt (1 mmol) of the product was dissolved in deionized water (20 mL), and then an aqueous solution of ammonium hexafluorophosphate (4 mmol) was added dropwise and reacted for 30 min, resulting in the precipitation of a light yellow solid. The mixture was filtered, washed with a large amount of deionized water, and dried in vacuo (60°C, 4 h) to obtain a hexafluorophosphate salt. The hexafluorophosphate salt was then dissolved in methanol (20 mL), and an aqueous solution of tetrabutylammonium chloride (4 mmol) was added dropwise and reacted for 30 min, resulting in the precipitation of a white solid. The mixture was filtered, washed with a large amount of ethyl acetate, and dried in vacuo (60°C, 4 h) to obtain a white powdery bipyridinium salt diphenylanthracene derivative PyEn (0.264 g, 80% yield).

[0059] S2. Preparation of water-soluble column [5] aromatic hydrocarbon (WP5C5), the reaction equation is as follows:

[0060]

[0061] S201. Hydroquinone (2.20 g, 20.0 mmol) and potassium carbonate (16.5 g, 120 mmol) were mixed in anhydrous acetonitrile (250 mL). After stirring at room temperature for 30 min, ethyl 5-bromovalerate (7.0 mL, 44.0 mmol) was added to the reaction system to carry out a nucleophilic substitution reaction. The reaction was refluxed at 100° C. for 48 h. After the reaction solution was cooled to room temperature, it was filtered, washed with water and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (volume ratio of petroleum ether and ethyl acetate was 10:1) to obtain product 4 as a white powder (6.10 g, yield 83%).

[0062] S202, product four (0.733 g, 2.00 mmol), paraformaldehyde (0.186 g, 6.00 mmol) and dichloroethane (100 mL) were mixed, and then boron trifluoride etherate (0.25 mL, 2.00 mmol) was added to carry out polymerization reaction. It can be observed that the reaction system gradually changed from colorless to dark green. After the reaction for 30 minutes, water (50 mL) was added to quench the reaction. The organic phase was then washed with water, sodium bicarbonate aqueous solution and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed in vacuo and then purified by silica gel column chromatography (the volume ratio of petroleum ether and ethyl acetate was 3:1) to obtain product five as a white powder (85.0 mg, yield 21%).

[0063] S203, tetrahydrofuran and water were mixed in a volume ratio of 1:1 to obtain 10 mL of a mixed solvent, followed by the addition of potassium hydroxide (0.168 g, 3.00 mmol) and product five (0.189 g, 0.100 mmol), mixed evenly, and hydrolyzed at 60°C for 12 h. After the reaction system was cooled to room temperature, the pH value of the reaction system was adjusted to 2 with 1.0 mM dilute hydrochloric acid. White flocculent material appeared, which was filtered off and washed with water several times. After vacuum drying (60°C, 4 h), a white powdery product six (0.178 g, 85% yield) was obtained.

[0064] S204, product VI (120 mg, 0.0749 mmol), potassium hydroxide (42.0 mg, 0.749 mmol) and water (5 mL) were mixed, stirred for 1 h, and then the solvent was removed in vacuo to obtain a white powder of water-soluble pillar [5] aromatic hydrocarbon WP5C5 (0.148 g, 100% yield);

[0065] S3. Bipyridinium diphenylanthracene derivative (PyEn) and water-soluble pillar[5]arene (WP5C5) were mixed in water at a molar ratio of 1:1 and stirred for 12 h to obtain supramolecular assembly SNPs.

[0066] Example 2

[0067] A method for preparing a supramolecular assembly responsive to spermine comprises the following steps:

[0068] S1. Preparation of bipyridinium diphenylanthracene derivatives (PyEn);

[0069] S101. 9,10-dibromoanthracene (12 mmol), 4-methoxy-2-methylphenylboronic acid (12 mmol), tetrakistriphenylphosphine palladium (3 mmol) and sodium carbonate (40 mL, 2 mol / L) were mixed in 100 mL of an organic solvent (the volume ratio of ethanol to toluene was 1:3), and the mixture was stirred at 110°C under an argon atmosphere for 12 h. After the reaction solution was cooled to room temperature, it was filtered, the filtrate was collected, and the solvent was removed by distillation under reduced pressure. The obtained crude product solid was dissolved in dichloromethane (80 mL), and after extraction with water, the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered again. The solvent was removed by distillation under reduced pressure, and then the mixture was flash chromatographed on a silica gel column (the volume ratio of dichloromethane to petroleum ether was 1:1) to obtain product 1 as a white powder.

[0070] S102, product 1 (780 mg, 1.86 mmol) was dissolved in anhydrous dichloromethane (50 mL), and boron tribromide (0.5 mL) was added dropwise to the reaction system under argon protection and ice bath conditions to carry out demethylation reaction. After 8 h of reaction, deionized water (20 mL) was added to quench the reaction, and a light yellow solid precipitated. The solid was filtered and washed with methanol, and the filter cake was decompressed to remove the solvent to obtain product 2 as a light yellow powder;

[0071] S103, product 2 (300 mg, 0.769 mmol), 1,6-dibromohexane (1.86 g, 7.69 mmol), anhydrous potassium carbonate (1.06 g, 7.69 mmol) and acetone (30 mL) were mixed uniformly, and then a nucleophilic substitution reaction was carried out at 90° C. under an argon atmosphere for 10 h. After the reaction solution was cooled to room temperature, potassium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to remove the solvent. The obtained solid was dissolved in dichloromethane, and after extraction and separation with water, the organic phase was washed with saturated brine (30 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered again, and the solvent was distilled under reduced pressure. After passing through a silica gel column for flash column chromatography (the chromatographic solvent was petroleum ether), a white powder of product 3 was obtained;

[0072] S104. Product 3 (300 mg, 0.420 mmol) was mixed with pyridine (5 mL), and the mixture was reacted at 70° C. for 9 h. After the reaction solution was cooled to room temperature, the solvent was distilled off under reduced pressure, and the obtained solid crude product was washed with ethyl acetate to obtain the bromide salt of the product;

[0073] The bromide salt (1 mmol) of the product was dissolved in deionized water (20 mL), and then an aqueous solution of ammonium hexafluorophosphate (2 mmol) was added dropwise and reacted for 25 min, resulting in the precipitation of a light yellow solid. The mixture was filtered, washed with a large amount of deionized water, and dried in vacuo (60°C, 4 h) to obtain a hexafluorophosphate salt. The hexafluorophosphate salt was then dissolved in methanol (20 mL), and an aqueous solution of tetrabutylammonium chloride (5 mmol) was added dropwise and reacted for 25 min, resulting in the precipitation of a white solid. The mixture was filtered, washed with a large amount of ethyl acetate, and dried in vacuo (60°C, 4 h) to obtain 0.264 g of a white powdery bipyridinium salt diphenylanthracene derivative PyEn.

[0074] S2, preparation of water-soluble column [5] aromatic hydrocarbons (WP5C5);

[0075] S201. Hydroquinone (2.20 g, 20.0 mmol) and potassium carbonate (16.5 g, 120 mmol) were mixed in anhydrous acetonitrile (250 mL). After stirring at room temperature for 30 min, ethyl bromoacetate (7.0 mL, 44.0 mmol) was added to the reaction system to carry out a nucleophilic substitution reaction. The reaction was refluxed at 110° C. for 46 h. After the reaction solution was cooled to room temperature, it was filtered, washed with water and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (the volume ratio of petroleum ether and ethyl acetate was 10:1) to obtain product 4 as a white powder.

[0076] S202, product four (0.733 g, 2.00 mmol), paraformaldehyde (0.186 g, 6.00 mmol) and dichloroethane (100 mL) were mixed, and then boron trifluoride etherate (0.25 mL, 2.00 mmol) was added to carry out polymerization reaction. It can be observed that the reaction system gradually changed from colorless to dark green. After the reaction for 35 minutes, water (50 mL) was added to quench the reaction. The organic phase was then washed with water, sodium bicarbonate aqueous solution and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed in vacuo and then purified by silica gel column chromatography (the volume ratio of petroleum ether and ethyl acetate was 3:1) to obtain product five as a white powder;

[0077] S203, tetrahydrofuran and water were mixed in a volume ratio of 1:1 to obtain 10 mL of a mixed solvent, followed by the addition of potassium hydroxide (0.168 g, 3.00 mmol) and product five (0.189 g, 0.100 mmol), mixed evenly, and hydrolyzed at 70°C for 10 h. After the reaction system was cooled to room temperature, the pH value of the reaction system was adjusted to 1.5 with 1.0 mM dilute hydrochloric acid. White flocculent material appeared, which was filtered, and the filtrate was washed with water several times and vacuum dried (60°C, 4 h) to obtain product six as a white powder;

[0078] S204, product VI (120 mg, 0.0749 mmol), potassium hydroxide (42.0 mg, 0.749 mmol) and water (5 mL) were mixed, stirred for 1.5 h and then the solvent was removed in vacuo to obtain a white powdery water-soluble columnar [5] aromatic hydrocarbon WP5C5;

[0079] S3. Bipyridinium diphenylanthracene derivative (PyEn) and water-soluble pillar[5]arene (WP5C5) were mixed in water at a molar ratio of 1:6, and stirred for 14 hours to obtain supramolecular assembly SNPs.

[0080] Example 3

[0081] A method for preparing a supramolecular assembly responsive to spermine comprises the following steps:

[0082] S1. Preparation of bipyridinium diphenylanthracene derivatives (PyEn);

[0083] S101. 9,10-dibromoanthracene (1.00 g, 2.99 mmol), 4-methoxy-2-methylphenylboronic acid (1.00 g, 2.99 mmol), dichlorobis(triphenylphosphine)palladium(II) (0.702 g, 0.608 mmol) and sodium carbonate (10 mL, 2 mol / L) were mixed in 40 mL of an organic solvent (the volume ratio of ethanol to toluene was 1:3), and the mixture was stirred at 120°C under a nitrogen atmosphere for 8 h. After the reaction solution was cooled to room temperature, it was filtered, the filtrate was collected, and the solvent was removed by distillation under reduced pressure. The obtained crude product solid was dissolved in dichloromethane (80 mL), extracted with water, and the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered again. The solvent was removed by distillation under reduced pressure, and the mixture was flash chromatographed on a silica gel column (the volume ratio of dichloromethane to petroleum ether was 1:1) to obtain product 1 as a white powder.

[0084] S102, product 1 (780 mg, 1.86 mmol) was dissolved in anhydrous dichloromethane (50 mL), and boron tribromide (0.5 mL) was added dropwise to the reaction system under argon protection and ice bath conditions to carry out demethylation reaction. After 12 h of reaction, deionized water (20 mL) was added to quench the reaction, and a light yellow solid was precipitated. The solid was filtered and washed with methanol, and the filter cake was decompressed to remove the solvent to obtain product 2 as a light yellow powder;

[0085] S103, product 2 (300 mg, 0.769 mmol), 1,6-dibromohexane (1.86 g, 7.69 mmol), anhydrous potassium carbonate (1.06 g, 7.69 mmol) and acetone (30 mL) were mixed uniformly, and then a nucleophilic substitution reaction was carried out under an argon atmosphere at 70°C for 14 h. After the reaction solution was cooled to room temperature, potassium carbonate was removed by filtration, and the filtrate was distilled under reduced pressure to remove the solvent. The obtained solid was dissolved in dichloromethane, and after extraction and separation with water, the organic phase was washed with saturated brine (30 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered again, and the solvent was distilled under reduced pressure. After passing through a silica gel column for flash column chromatography (the chromatographic solvent was petroleum ether), a white powder of product 3 was obtained;

[0086] S104. Product 3 (300 mg, 0.420 mmol) was mixed with pyridine (5 mL), and the mixture was reacted at 90° C. for 7 h. After the reaction solution was cooled to room temperature, the solvent was removed by distillation under reduced pressure. The obtained solid crude product was washed with ethyl acetate to obtain a bromide salt of the product.

[0087] The bromide salt (1 mmol) of the product was dissolved in deionized water (20 mL), and then an aqueous solution of ammonium hexafluorophosphate (2 mmol) was added dropwise and reacted for 35 min, resulting in the precipitation of a light yellow solid. The mixture was filtered, washed with a large amount of deionized water, and dried in vacuo (60°C, 4 h) to obtain a hexafluorophosphate salt. The hexafluorophosphate salt was then dissolved in methanol (20 mL), and an aqueous solution of tetrabutylammonium chloride (5 mmol) was added dropwise and reacted for 35 min, resulting in the precipitation of a white solid. The mixture was filtered, washed with a large amount of ethyl acetate, and dried in vacuo (60°C, 4 h) to obtain a white powdery bipyridinium salt diphenylanthracene derivative PyEn.

[0088] S2, preparation of water-soluble column [5] aromatic hydrocarbons (WP5C5);

[0089] S201. Hydroquinone (2.20 g, 20.0 mmol) and potassium carbonate (16.5 g, 120 mmol) were mixed in anhydrous acetonitrile (250 mL). After stirring at room temperature for 30 min, ethyl 5-bromovalerate (7.0 mL, 44.0 mmol) was added to the reaction system to carry out a nucleophilic substitution reaction. The reaction was refluxed at 90° C. for 50 h. After the reaction solution was cooled to room temperature, it was filtered, washed with water and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (the volume ratio of petroleum ether and ethyl acetate was 10:1) to obtain product 4 as a white powder.

[0090] S202, product four (0.733 g, 2.00 mmol), paraformaldehyde (0.186 g, 6.00 mmol) and dichloroethane (100 mL) were mixed, and then boron trifluoride etherate (0.25 mL, 2.00 mmol) was added to carry out polymerization reaction. It can be observed that the reaction system gradually changed from colorless to dark green. After reacting for 25 minutes, water (50 mL) was added to quench the reaction. The organic phase was then washed with water, sodium bicarbonate aqueous solution and saturated brine in sequence, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed in vacuo and then purified by silica gel column chromatography (the volume ratio of petroleum ether and ethyl acetate was 3:1) to obtain product five as a white powder;

[0091] S203, tetrahydrofuran and water were mixed in a volume ratio of 1:1 to obtain 10 mL of a mixed solvent, followed by the addition of potassium hydroxide (0.168 g, 3.00 mmol) and product five (0.189 g, 0.100 mmol), mixed evenly, and hydrolyzed at 50°C for 14 h. After the reaction system was cooled to room temperature, the pH value of the reaction system was adjusted to 2.5 with 1.0 mM dilute hydrochloric acid. White flocculent material appeared, which was filtered, and the filtrate was washed with water several times and vacuum dried (60°C, 4 h) to obtain product six as a white powder;

[0092] S204, product VI (120 mg, 0.0749 mmol), potassium hydroxide (42.0 mg, 0.749 mmol) and water (5 mL) were mixed, stirred for 0.5 h and then the solvent was removed in vacuo to obtain a white powdery water-soluble columnar [5] aromatic hydrocarbon WP5C5;

[0093] S3. The bipyridinium diphenylanthracene derivative (PyEn) and water-soluble pillar[5]arene (WP5C5) were mixed in water at a molar ratio of 1:0.5, stirred and reacted for 10 hours to obtain supramolecular assembly SNPs.

[0094] The following experiments were carried out using the supramolecular assembly SNPs prepared in Example 1.

[0095] Experimental Example 1 Structural Characterization

[0096] Figure 1 The particle size distribution diagram of the supramolecular assembly is shown in Figure 2. From the figure, we can see that the particle size of the supramolecular assembly is about 140nm. The transmission electron microscopy morphology of the supramolecular assembly is shown in Figure 2. Figure 2 As shown, it is shown that the supramolecular assembly can be successfully prepared by the method of the present invention.

[0097] Dynamic light scattering experiments were used to monitor the changes in particle size and polydispersity index (PDI) of the supramolecular assembly SNPs in water to evaluate the stability of the supramolecular assembly. Figure 3It can be seen that the particle size and polydispersity index (PDI) of the supramolecular assembly SNPs remain substantially unchanged, and the supramolecular assembly SNPs prepared in the present invention are stable in water.

[0098] Experimental Example 2 Evaluation of Sexual Release Performance

[0099] The release behavior of PyEn was monitored by H NMR spectroscopy: First, the H NMR spectrum of the supramolecular assembly SNPs in heavy water (WP5C5 / PyEn=2 / 1, PyEn concentration was 2.5mM / L) was recorded. Then, 0 to 2 equivalents of spermine were added to the supramolecular assembly SNPs solution, and the changes in the H NMR spectrum were recorded by H NMR spectroscopy. The results are shown in Figure 2. Figure 4 shown.

[0100] The release behavior of PyEn was monitored by fluorescence spectroscopy: First, the fluorescence intensity of the supramolecular assembly SNPs in water was recorded (WP5C5 / PyEn=2 / 1, the concentration of PyEn was 0.01mM / L), and then 0-3 equivalents of spermine were added to the supramolecular assembly SNPs solution, and the changes in fluorescence emission intensity were recorded by fluorescence spectrometer. The results are shown in Figure 2. Figure 5 shown.

[0101] Depend on Figure 4 and Figure 5 It can be seen that the supramolecular assembly SNPs prepared in the present invention has obvious spermine competitiveness and has the property of being used as an anthracycline anti-tumor agent.

[0102] Experimental Example 3 Evaluation of cancer cell uptake behavior

[0103] In order to evaluate the uptake of therapeutic nanoparticles by cancer cells, A549 cancer cells were co-incubated with the supramolecular assembly for 2-6 hours and then laser confocal microscopy was performed. The specific process was as follows: A549 cells were first seeded in a 35mm cell immunofluorescence dish, and when they grew to a suitable density, The cells were incubated for 2, 4, and 6 hours, followed by aspiration of the culture medium and washing three times with PBS before observation under a laser confocal microscope. Imaging conditions: PyEn: excitation wavelength 405 nm, emission wavelength 420-500 nm.

[0104] The results are as follows Figure 6 As shown, the supramolecular assembly can be taken up by cancer cells after 2 h of co-incubation.

[0105] Experimental Example 4: Subcellular organelle targeting evaluation

[0106] To evaluate To investigate the mitochondrial and lysosomal targeting capabilities of the nanoparticles, A549 cancer cells were incubated with the nanoparticles for 4 hours in a confocal microplate. Mitochondrial probes (Mito-tracker Red) (2 μM) or lysosomal probes (Lysol-tracker Red) (2 μM) were then added and incubated for another 30 minutes before confocal microscopy. Imaging conditions: PyEn: excitation wavelength 405 nm, emission wavelength 420-500 nm; Mito-tracker Red: excitation wavelength 552 nm, emission wavelength 600-650 nm; Lysol-tracker Red: excitation wavelength 552 nm, emission wavelength 600-650 nm.

[0107] The results are as follows Figure 7 As shown, there is a large overlap between the fluorescence of the anthracene ring in the supramolecular assembly and the fluorescence of the mitochondrial probe, which proves that the supramolecular assembly SNPs prepared by the present invention has good mitochondrial targeting.

[0108] Experimental Example 5: Evaluation of cytotoxicity in normal cells

[0109] To evaluate the cytotoxicity of SNPs to normal cells, HK2 cells were plated at 5 × 10 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured overnight. Subsequently, the cells were incubated with various concentrations of WP5C5 in an incubator for 24 hours. The culture medium was then removed, the cells were washed three times with PBS, and CCK-8 solution (10 μL) and fresh culture medium (90 μL) were added to each well for a further 1 hour. Finally, the absorbance at 450 nm was measured using a microplate reader. Relative cell viability was calculated using cells treated with culture medium as a control group.

[0110] The results are as follows Figure 8 As shown, it is shown that the supramolecular assembly SNPs prepared by the present invention has good biocompatibility to normal cells.

[0111] Experimental Example 6 Evaluation of cytotoxicity in cancer cells overexpressing spermine

[0112] To evaluate the cytotoxicity of SNPs against spermine-overexpressing cancer cells, A549 cells were plated at 5 × 10 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured overnight. The cells were then incubated with various concentrations of WP5C5 in an incubator for 24 hours. The culture medium was removed, the cells were washed three times with PBS, and CCK-8 solution (10 μL) and fresh culture medium (90 μL) were added to each well for a further 1 hour. Finally, the absorbance at 450 nm was measured using a microplate reader. Relative cell viability was calculated using cells treated with culture medium as a control group.

[0113] The results are as follows Figure 9 and Figure 10As shown, the supramolecular assembly exhibited high cytotoxicity against cancer cells.

[0114] Experimental Example 7 Evaluation of the Inhibition of Therapeutic Nanoparticles on Solid Tumors in Mice

[0115] A549 / ADR tumor-bearing mice were used as animal models for in vivo anti-tumor research. The specific research process was as follows: when the tumor volume of the mouse reached 10 mm 3 The mice were randomly divided into three groups (5 mice in each group), namely PBS control group, PyEn and Treatment group: Mice were injected with drugs via the tail vein once every three days at a dose of 4 mg / kg (equivalent concentration of PyEn) for 13 days.

[0116] Depend on Figure 11 It can be seen that compared with the control group, the (i.e., supramolecular assembly SNPs) showed significant inhibition on tumor growth, and (SNPs) have a slightly stronger inhibitory effect on tumor growth than PyEn alone. This is because Stable supramolecular nanoparticles (SNPs) can be effectively enriched in tumor tissues through the EPR effect. Overexpression of SPM in A549 / ADR tumor cells promotes Effective release of PyEn in SNPs at tumor sites.

[0117] Depend on Figure 12 and Figure 13 It can be seen that the weight of mice in each treatment group remained basically unchanged during the treatment process, and the sections of the main organs in each group showed no obvious tissue lesions after treatment, indicating that (SNPs) have basically no toxic side effects under the experimental conditions and have good biosafety.

Claims

1. A method for preparing a supramolecular assembly responsive to spermine, characterized in that: The bipyridinium salt diphenylanthracene derivative and the water-soluble pillar[5]arene are self-assembled in water to obtain a supramolecular assembly, which specifically includes the following steps: S1. preparing a bipyridinium salt diphenylanthracene derivative; S101, mixing a diphenylanthracene compound, a boric acid compound, a palladium catalyst, and a base in an organic solvent at a molar ratio of 12-17:12-17:2-4:80-120, and reacting under a protective atmosphere at 110-120° C. for 8-12 hours to obtain product 1; S102, dissolving the product 1 in an organic solvent, and then adding a demethylation reagent to react for 8-12 hours to obtain the product 2; the ratio of the product 1 to the demethylation reagent is 3.5-4 mmol: 0.8-1.2 mL; S103, product 2, a bromine-containing compound, and a base are mixed in an organic solvent at a molar ratio of 1-2:10-20:10-20, and then reacted under a protective atmosphere at 70-90° C. for 10-14 hours to obtain product 3; S104, mixing the product three with pyridine in a ratio of 0.3-0.5 mmol: 3-7 mL, and reacting at 70-90° C. for 7-9 hours to obtain a crude product; mixing the crude product with an ammonium hexafluorophosphate solution for reaction for 20-40 minutes, filtering, drying, and mixing the dried product with a tetrabutylammonium chloride solution for reaction for 20-40 minutes to obtain a bipyridinium salt diphenylanthracene derivative; the molar ratio of the crude product, the ammonium hexafluorophosphate solution, and the tetrabutylammonium chloride solution is 1:2-5:2-5; S2, preparing water-soluble column [5] aromatic hydrocarbons; S3. Mixing a bipyridinium salt diphenylanthracene derivative and a water-soluble pillar[5]arene at a molar ratio of 1:0.5-6 and reacting for 10-14 hours to obtain a supramolecular assembly.

2. The method for preparing a spermine-responsive supramolecular assembly according to claim 1, wherein: The diphenylanthracene compound is 9,10-dibromoanthracene; the boronic acid compound is 4-methoxy-2-methylphenylboronic acid; the palladium catalyst is tetrakistriphenylphosphine palladium or dichlorobis(triphenylphosphine)palladium(II); and the base is sodium carbonate, potassium carbonate or potassium hydroxide.

3. The method for preparing a spermine-responsive supramolecular assembly according to claim 1, wherein: The protective atmosphere is argon or nitrogen.

4. The method for preparing a supramolecular assembly responsive to spermine according to claim 1, wherein The demethylation reagent is boron tribromide.

5. The method for preparing a supramolecular assembly responsive to spermine according to claim 1, wherein: The bromine-containing compound is 1,6-dibromohexane, ethyl 5-bromovalerate or ethyl bromoacetate.

6. The method for preparing a supramolecular assembly responsive to spermine according to claim 1, wherein: The molar ratio of the bipyridinium diphenylanthracene derivative and the water-soluble pillar [5] aromatic hydrocarbon in S3 is 1:

1.

7. A supramolecular assembly responsive to spermine, characterized in that: The method is prepared according to any one of claims 1 to 6.

8. Use of the spermine-responsive supramolecular assembly according to claim 7 in the preparation of drugs for treating tumors.

9. The use according to claim 8, characterized in that The tumor treatment drug is an injectable drug.

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