A Matrine Derivative Nanoparticle and Its Preparation Method and Application

Abstract

The present invention discloses a matrine derivative nanoparticle, a preparation method and an application thereof, belonging to the technical field of medicine. Based on the structural modification of matrine, the present invention synthesizes a new matrine derivative (LST-4). On this basis, nanoparticles (LST-4 NPs) are further self-assembled. The reaction conditions of the present invention are mild, and the particle size of the nanoparticles is uniform and stable. The matrine derivative nanoparticle LST-4 NPs of the material can smoothly enter cells and inhibit the proliferation of tumor cells. After entering cells, LST-4 NPs aggregate in lysosomes, improving the bioavailability of the drug; compared with LST-4, LST-4 NPs enhance the EPR effect, and in the aspect of tumor treatment, LST-4 NPs have better treatment effects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a matrine derivative nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Natural products have extensive biological activities and low toxicity, and thus are widely studied as anti-cancer drugs. Matrine is one of the key tetracyclic quinoline alkaloids isolated from the roots of Sophora flavescens, and has a variety of pharmacological effects, such as sedation, anti-inflammatory, immunomodulatory, antiviral, and anti-tumor. Large-scale applications of matrine include its use in the treatment of cancer. A number of studies have confirmed that matrine has anti-cancer activity against gastric cancer, liver cancer, leukemia K-562, lung cancer, and cervical cancer. Previous studies have confirmed that matrine can inhibit the proliferation and migration of HepG 2 liver cancer cells. In addition, the inhibitory effect of matrine on liver cancer has been applied clinically. Some in vitro studies suggest that different matrine derivatives have specificity for different liver cancer cells, and the deeper mechanism may be achieved through the extracellular regulated protein kinase (ERK) signaling pathway.

[0003] However, matrine has obvious hepatotoxicity and neurotoxicity in vivo and in vitro. In addition, its bioavailability is relatively low. Nanoparticles are a kind of submicron drug delivery system prepared by adsorbing or encapsulating drugs, and have advantages such as high drug loading capacity, good stability, and biodegradability. A large number of studies have shown that nanoemulsions can significantly improve the dissolution performance of drugs, maintain drug stability, enhance body absorption, and thus improve their oral bioavailability. In addition, nanoformulations for drug delivery also have targeting and sustained release effects, which can achieve the effect of enhancing efficacy and reducing toxicity. Nanoemulsion drug delivery systems have been favored by researchers in the field of pharmaceutical preparations in recent years. As an emerging technology with great market potential, it has broad application prospects. Therefore, the preparation of matrine-based nano-drugs is of great significance for anti-tumor research. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides a matrine derivative nanoparticle, a preparation method thereof, and an application thereof. Since the nanoparticles have uniform and stable particle sizes, they can smoothly enter cells and inhibit the proliferation of tumor cells.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A matrine derivative, the structural formula of which is:

[0007]

[0008] The present invention also provides a preparation method of the matrine derivative as described above, comprising the following steps:

[0009] 1) Dissolve pentaethylene glycol in dichloromethane, then successively add p-toluenesulfonyl chloride and potassium hydroxide powder. After the reaction is completed, extract and remove impurities to obtain Compound 1 (3,6,9,12-tetraoxa-1,14-di(4-methyl)benzenesulfonate tetradecane);

[0010] 2) Add sodium azide to Compound 1 and carry out an oil bath reaction to obtain Compound 2 (1,14-diazido-3,6,9,12-tetraoxatetradecane);

[0011] 3) Mix Compound 2 with triphenylphosphine, react at room temperature, adjust the pH to 14 and then extract to obtain Compound 3 (14-diazido-3,6,9,12-tetraoxatetradecylamine);

[0012] 4) Add thalidomide, N,N-diisopropylethylamine and N,N-dimethylformamide to Compound 3 and carry out an oil bath reaction to obtain Compound 4 (4-((14-diazido-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxo-3-piperidinyl)isoindole-1,3-dione);

[0013] 5) Using matrine as the raw material and lithium diisopropylamide (LDA) as the base, remove the α-position hydrogen of the amide in matrine, and then directly carry out a nucleophilic substitution reaction with diphenyl disulfide to obtain Compound 14-phenylthio matrine. After oxidation with iodobenzene diacetate (IBX) in the aqueous phase, Compound 14-phenylsulfinyl matrine is obtained. Under the action of potassium carbonate, benzenesulfinic acid is eliminated to obtain Compound 5 (sophocarpine);

[0014] Dissolve Compound 5 and glycine in water, heat and react, and recrystallize to obtain Compound 6 (15-aminoacetate matrine);

[0015] Mix methyl Boc-L-tyrosinate, N,N-dimethylformamide solution and potassium carbonate, stir at room temperature, add propargyl bromide, react at room temperature, and carry out extraction after the reaction is completed to obtain Compound 7 (methyl 2-((tert-butoxycarbonyl)amino)-3-(4-(2-propynyloxy)phenyl)propionate);

[0016] Place Compound 7 in a hydrochloric acid ethyl acetate solution, stir at room temperature overnight, remove the solvent after the reaction is completed to obtain Compound 8 (methyl 2-amino-3-(4-(2-propynyloxy)phenyl)propionate);

[0017] Dissolve Compound 6 and Compound 8 in anhydrous dichloromethane, successively add EDCI, HOBt and DIPEA, carry out extraction and impurity removal after the reaction is completed at room temperature to obtain Compound 9 (15-((2-(2-aminoacetate)amino)-3-(4-(2-propynyloxy)phenyl)propionate methyl)matrine);

[0018] 6) Dissolve Compound 4 and Compound 9 in a tetrahydrofuran solution, stir at room temperature to obtain a reaction solution;

[0019] Dissolve copper sulfate and sodium ascorbate in water. After shaking well, the color of the solution turns brown;

[0020] Drop the brown aqueous solution into the reaction solution and stir at room temperature to obtain matrine derivative LST-4.

[0021] The present invention also provides a matrine derivative nanoparticle, which uses the matrine derivative described above to self-assemble into spherical nano-molecules, namely matrine derivative nanoparticles.

[0022] The present invention also provides a preparation method of the matrine derivative nanoparticle, including the following steps: dissolve the matrine derivative in acetonitrile, inject the obtained solution into ultrapure water, and perform ultrasonic treatment under a nitrogen atmosphere to obtain the matrine derivative nanoparticle.

[0023] The present invention also provides an application of the matrine derivative nanoparticle described above in the preparation of anti-tumor drugs.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] The reaction conditions of the present invention are mild, and the particle size of the nanoparticles is uniform and stable. The material, matrine derivative nanoparticle LST-4 NPs, can smoothly enter cells and inhibit the proliferation of tumor cells. After LST-4 NPs enter cells, they aggregate in lysosomes, improving the bioavailability of the drug; compared with LST-4, LST-4 NPs enhance the EPR effect, and in tumor treatment, LST-4 NPs have better treatment effects. Description of the Drawings

[0026] The drawings constituting a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application. In the drawings:

[0027] Figure 1 is the ultraviolet-visible spectrophotometric analysis of LST-4;

[0028] Figure 2 is the linear relationship diagram of the absorbance at different concentrations of LST-4 varying with concentration at the maximum absorption wavelength (λ max ) in the wavelength range of 250 to 350 nm;

[0029] Figure 3 is the fluorescence spectral analysis of LST-4;

[0030] Figure 4is the linear relationship diagram of the fluorescence intensity of LST-4 at different concentrations with the change of concentration at the emission wavelength (λ em );

[0031] Figure 5 is the visualized fluorescence imaging diagram of LST-4 NPs in vitro;

[0032] Figure 6 is the relative fluorescence intensity of LST-4 NPs at different concentrations;

[0033] Figure 7 is the TEM analysis of LST-4 NPs;

[0034] Figure 8 is the particle size analysis of LST-4 NPs;

[0035] Figure 9 is the potential analysis of LST-4 NPs;

[0036] Figure 10 is the cytotoxicity of LST-4;

[0037] Figure 11 is the cytotoxicity of LST-4 NPs;

[0038] Figure 12 is the cellular uptake of LST-4 NPs;

[0039] Figure 13 is the subcellular localization of LST-4 NPs;

[0040] Figure 14 is the schematic diagram of nano self-assembly of LST-4 NPs. Detailed implementation manners

[0041] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0043] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0044] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of this invention will be obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0045] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0046] As used in this invention, "room temperature" is calculated as 25 ± 2 °C unless otherwise specified.

[0047] All raw materials used in the following examples of this invention are obtained commercially.

[0048] A matrine derivative, the structural formula of which is:

[0049]

[0050] The matrine derivative LST-4 is prepared according to the following reaction route:

[0051]

[0052]

[0053] The specific synthesis route of LST-4 is as follows:

[0054] 1) Under the condition of an ice bath at 0 °C, dissolve pentaethylene glycol (3.57 g, 15 mmol, 1 eq) in dichloromethane (100 mL). After complete dissolution, add p-toluenesulfonyl chloride (5.72 g, 30 mmol, 2 eq), stir evenly, then add powdered potassium hydroxide (6.72 g, 120 mmol, 8 eq), and react for 3 h. After the reaction is completed, add (100 mL) ice water to the system, and then add dichloromethane for extraction. The organic layer is dried with anhydrous sodium sulfate. After standing, the solvent is evaporated to dryness using a rotary evaporator to obtain Compound 1.

[0055] 2) Dissolve compound 1 (5.64 g, 10 mmol, 1 eq) in 20 mL of DMF, stir until completely dissolved, add sodium azide (1.43 g, 22 mmol, 2.2 eq), and react at 60 °C in an oil bath for 4 h. After monitoring the completion of the reaction by TLC, add 20 mL of water to quench the reaction, extract the reaction mixture with ethyl acetate, wash the organic layer once with saturated brine, then add anhydrous sodium sulfate for drying, and let it stand overnight. Evaporate the solvent using a rotary evaporator to obtain compound 2.

[0056] 3) Add an anhydrous ether∶tetrahydrofuran∶1 mol / L hydrochloric acid (10 mL∶8 mL∶2 mL) solution to compound 2 (2.30 g, 8 mmol, 1 eq), then weigh triphenylphosphine (2.14 g, 8 mmol, 1 eq) and slowly add it thereto, stir, and react at room temperature for 12 h. After monitoring the completion of the reaction by TLC, add 4 mol / L hydrochloric acid until the solution is acidic (pH = 6). Wash the reaction mixture with ethyl acetate, add sodium hydroxide to the aqueous layer until pH = 14, then extract the compound with dichloromethane again. Wash the organic layer once with saturated brine. Then add anhydrous sodium sulfate for drying and let it stand overnight. Evaporate the solvent using a rotary evaporator to obtain compound 3.

[0057] 4) Add thalidomide (0.83 g, 3 mmol, 1 eq), N,N-diisopropylethylamine (0.66 g, 5.1 mmol, 1.7 eq), and 8 mL of N,N-dimethylformamide to compound 3 (0.79 g, 3 mmol, 1 eq), stir, and react in an oil bath at 90 °C for 4 h. After monitoring the completion of the reaction by TLC, add 8 mL of water to quench the reaction, extract the reaction mixture with ethyl acetate, wash the organic layer once with saturated brine, then add anhydrous sodium sulfate for drying, and let it stand overnight. Evaporate the solvent using a rotary evaporator, concentrate, and purify by silica gel column chromatography. The eluent is petroleum ether and ethyl acetate, and the volume ratio of the two is 6∶1 to obtain compound 4.

[0058] 5) Add 70 mL of tetrahydrofuran, 5.0 mL of diisopropylamine, and 16.1 mL (38.9 mmol) of a n-hexane solution of n-butyllithium (2.4 mol / L) to a flask. Under argon protection, the temperature is -78 °C. After stirring for 15 min, add 4.0 g (16.1 mmol) of matrine and a tetrahydrofuran solution (30 mL) of 3.6 g (16.4 mmol) of diphenyldisulfide to the flask. Raise the temperature to room temperature, stir for 2 h, then add 50 mL of saturated sodium carbonate solution, separate the layers, extract the aqueous phase with ethyl acetate, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and remove the solvent to obtain compound 5.

[0059] 6) Dissolve compound 5 (0.31 g, 1.25 mmol, 1 eq) and glycine (0.19 g, 2.5 mmol, 2 eq) in water (2.0 mL), stir, and react at 80 °C for 12 h. After monitoring the completion of the reaction by TLC, evaporate the solvent under reduced pressure. Recrystallize with an ethanol-water system to obtain compound 6.

[0060] 7) Dissolve Boc-L-tyrosine methyl ester (5.90 g, 20 mmol, 1 eq) in 15 mL of N,N-dimethylformamide solution. After dissolution, add potassium carbonate (5.53 g, 40 mmol, 2 eq), stir at room temperature for 10 min, then add propargyl bromide (4.76 g, 40 mmol, 2 eq), and react at room temperature for 6 h. After monitoring the end of the reaction by TLC, extract with 200 mL of saturated sodium chloride solution and 200 mL of ethyl acetate solution. Dry the organic layer with anhydrous sodium sulfate, concentrate by standing, and purify by silica gel column chromatography. The eluent is petroleum ether and ethyl acetate, and the volume ratio of the two is 5:1 to obtain compound 7.

[0061] 8) Place compound 7 in a hydrochloric acid-ethyl acetate solution (volume ratio 1:1), stir at room temperature overnight to remove the Boc group. Detect the reaction result by thin layer chromatography. After the reaction is completed, remove the solvent to obtain compound 8 as a white solid, which is directly used for the next step of the reaction.

[0062] 9) Under the condition of 0 °C, dissolve compound 6 (0.32 g, 1 mmol, 1 eq) and compound 8 (0.23 g, 1 mmol, 1 eq) in 50 mL of anhydrous dichloromethane. After dissolution, sequentially add EDCI (0.23 g, 1.2 mmol, 1.2 eq), HOBt (0.16 g, 1.2 mmol, 1.2 eq), and DIPEA (0.16 g, 1.2 mmol, 1 eq). After 20 min, place the reaction at room temperature and stir overnight. Detect the reaction result by thin layer chromatography. After the reaction is completed, add 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate aqueous solution to the reaction solution for extraction and removal of impurities. Wash the organic phase with 50 mL of saturated sodium chloride aqueous solution, and dry the organic layer with anhydrous sodium sulfate. After standing, remove the solvent with a rotary evaporator. Purify by silica gel column chromatography, and the eluent is dichloromethane and methanol with a volume ratio of 20:1 to obtain the corresponding amide product, which is compound 9.

[0063] 10) Compound 4 (0.26 g, 0.5 mmol, 1 eq) and compound 9 (0.27 g, 0.5 mmol, 1 eq) were dissolved in 2 mL of tetrahydrofuran solution, and the reaction mixture was stirred at room temperature. Copper sulfate (0.15 g, 0.5 mmol, 1 eq) and sodium ascorbate (0.20 g, 1 mmol, 2 eq) were dissolved in 0.5 mL of water. After shaking well, the color of the solution turned brown. The brown aqueous solution was added dropwise to the tetrahydrofuran reaction solution, and the mixture was stirred at room temperature for 10 h. The reaction result was detected by thin layer chromatography. After the reaction was completed, the solvent was removed by a rotary evaporator. Purification by silica gel column chromatography was carried out with dichloromethane and methanol as the eluent, and the volume ratio was 15:1, to obtain a yellow solid, that is, the product matrine derivative LST-4.

[0064] The present invention also provides a matrine derivative nanoparticle, which uses the matrine derivative described above to self-assemble into spherical nano-molecules, that is, matrine derivative nanoparticle LST-4NPs.

[0065] The above LST-4 and LST-4NPs have cytotoxic effects on HepG 2 cells, and it is found that LST-4NPs can significantly enhance the toxic effect on HepG 2.

[0066] The present invention also provides a preparation method of a matrine derivative nanoparticle, which includes the following steps: dissolving the matrine derivative in acetonitrile as a mother liquor, and slowly injecting the mother liquor into ultrapure water by a pipette gun, and performing ultrasonic treatment under a nitrogen atmosphere to obtain the matrine derivative nanoparticle.

[0067] The present invention also provides an application of the matrine derivative nanoparticle described above in the preparation of anti-tumor drugs.

[0068] The following examples are further descriptions of the technical solutions of the present invention.

[0069] Example 1

[0070] Synthesis of LST-4:

[0071] 1) Under the condition of an ice bath at 0 °C, pentaerythritol (3.57 g, 15 mmol, 1 eq) was dissolved in dichloromethane (100 mL). After complete dissolution, p-toluenesulfonyl chloride (5.72 g, 30 mmol, 2 eq) was added, and the mixture was stirred evenly. Then, powdered potassium hydroxide (6.72 g, 120 mmol, 8 eq) was added, and the reaction was carried out for 3 h. After the reaction was completed, 100 mL of ice water was added to the system, and then dichloromethane was added for extraction. The organic layer was dried with anhydrous sodium sulfate. After standing, the solvent was evaporated to dryness using a rotary evaporator to obtain compound 1.

[0072] 2) Dissolve compound 1 (5.64 g, 10 mmol, 1 eq) in 20 mL of DMF, stir until completely dissolved, add sodium azide (1.43 g, 22 mmol, 2.2 eq), and react at 60 °C in an oil bath for 4 h. After monitoring the completion of the reaction by TLC, add 20 mL of water to quench, extract the reaction mixture with ethyl acetate, wash the organic layer once with saturated brine, then add anhydrous sodium sulfate for drying, and let it stand overnight. Evaporate the solvent using a rotary evaporator to obtain compound 2.

[0073] 3) Add anhydrous ether∶tetrahydrofuran∶1 mol / L hydrochloric acid (10 mL∶8 mL∶2 mL) solution to compound 2 (2.30 g, 8 mmol, 1 eq), then weigh triphenylphosphine (2.14 g, 8 mmol, 1 eq) and slowly add it thereto, stir, and react at room temperature for 12 h. After monitoring the completion of the reaction by TLC, add 4 mol / L hydrochloric acid until the solution is acidic (pH = 6). Wash the reaction mixture with ethyl acetate, add sodium hydroxide to the aqueous layer until pH = 14, then extract the compound with dichloromethane again, wash the organic layer once with saturated brine. Then add anhydrous sodium sulfate for drying, and let it stand overnight. Evaporate the solvent using a rotary evaporator to obtain compound 3.

[0074] 4) Add thalidomide (0.83 g, 3 mmol, 1 eq), N,N-diisopropylethylamine (0.66 g, 5.1 mmol, 1.7 eq), and 8 mL of N,N-dimethylformamide to compound 3 (0.79 g, 3 mmol, 1 eq), stir, and react in an oil bath at 90 °C for 4 h. After monitoring the completion of the reaction by TLC, add 8 mL of water to quench, extract the reaction mixture with ethyl acetate, wash the organic layer once with saturated brine, then add anhydrous sodium sulfate for drying, and let it stand overnight. Evaporate the solvent using a rotary evaporator, concentrate, and purify by silica gel column chromatography. The eluent is petroleum ether and ethyl acetate with a volume ratio of 6∶1 to obtain compound 4.

[0075] 5) Add 70 mL of tetrahydrofuran, 5.0 mL of diisopropylamine, and 16.1 mL (38.9 mmol) of a n-hexane solution of n-butyllithium (2.4 mol / L) to a flask. Under argon protection, the temperature is -78 °C. After stirring for 15 min, add 4.0 g (16.1 mmol) of matrine and a tetrahydrofuran solution (30 mL) of 3.6 g (16.4 mmol) of diphenyldisulfide to the flask. Raise the temperature to room temperature, stir for 2 h, then add 50 mL of saturated sodium carbonate solution, separate the layers, extract the aqueous phase with ethyl acetate, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and remove the solvent to obtain compound 5.

[0076] 6) Compound 5 (0.31 g, 1.25 mmol, 1 eq) and glycine (0.19 g, 2.5 mmol, 2 eq) were dissolved in water (2.0 mL), stirred, and reacted at 80 °C for 12 h. After monitoring the completion of the reaction by TLC, the solvent was evaporated under reduced pressure. Recrystallization was carried out using an ethanol - water system to obtain Compound 6.

[0077] 7) Boc - L - tyrosine methyl ester (5.90 g, 20 mmol, 1 eq) was dissolved in 15 mL of N,N - dimethylformamide solution. After dissolution, potassium carbonate (5.53 g, 40 mmol, 2 eq) was added, and the mixture was stirred at room temperature for 10 min. Then, propargyl bromide (4.76 g, 40 mmol, 2 eq) was added, and the reaction was carried out at room temperature for 6 h. After monitoring the end of the reaction by TLC, extraction was performed with 200 mL of saturated sodium chloride solution and 200 mL of ethyl acetate solution. The organic layer was dried over anhydrous sodium sulfate, concentrated by standing, and purified by silica gel column chromatography. The eluent was petroleum ether and ethyl acetate with a volume ratio of 5∶1 to obtain Compound 7.

[0078] 8) Compound 7 was placed in a hydrochloric acid - ethyl acetate solution (volume ratio 1:1) and stirred overnight at room temperature to remove the Boc group. The reaction result was detected by thin - layer chromatography. After the reaction was completed, the solvent was removed to obtain Compound 8 as a white solid, which was directly used for the next reaction.

[0079] 9) Under the condition of 0 °C, Compound 6 (0.32 g, 1 mmol, 1 eq) and Compound 8 (0.23 g, 1 mmol, 1 eq) were dissolved in 50 mL of anhydrous dichloromethane. After dissolution, EDCI (0.23 g, 1.2 mmol, 1.2 eq), HOBt (0.16 g, 1.2 mmol, 1.2 eq), and DIPEA (0.16 g, 1.2 mmol, 1 eq) were added in sequence. After 20 min, the reaction was stirred overnight at room temperature. The reaction result was detected by thin - layer chromatography. After the reaction was completed, 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate aqueous solution were added to the reaction solution for extraction and removal of impurities. The organic phase was washed with 50 mL of saturated sodium chloride aqueous solution, and the organic layer was dried over anhydrous sodium sulfate. After standing, the solvent was removed using a rotary evaporator. Purification was carried out by silica gel column chromatography. The eluent was dichloromethane and methanol with a volume ratio of 20∶1 to obtain the corresponding amide product, Compound 9.

[0080] (10) Compound 4 (0.26 g, 0.5 mmol, 1 eq) and compound 9 (0.27 g, 0.5 mmol, 1 eq) were dissolved in 2 mL of tetrahydrofuran solution, and the reaction mixture was stirred at room temperature. Copper sulfate (0.15 g, 0.5 mmol, 1 eq) and sodium ascorbate (0.20 g, 1 mmol, 2 eq) were dissolved in 0.5 mL of water. After shaking well, the color of the solution turned brown. The brown aqueous solution was added dropwise to the tetrahydrofuran reaction solution, and the mixture was stirred at room temperature for 10 h. The reaction result was detected by thin-layer chromatography. After the reaction was completed, the solvent was removed by a rotary evaporator. It was purified by silica gel column chromatography, and the eluent was dichloromethane and methanol with a volume ratio of 15:1 to obtain a yellow solid, that is, the product matrine derivative LST-4.

[0081] The structure of LST-4 was characterized by 1H-NMR and MS, and the results were as follows: 1 1H-NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.16 (d, J = 3.1 Hz, 1H), 7.57 (dd, J = 8.5, 7.1 Hz, 1H), 7.14–7.10 (m, 3H), 7.04 (d, J = 7.0 Hz, 1H), 6.95–6.91 (m, 2H), 6.59 (t, J = 5.8 Hz, 1H), 5.75 (s, 1H), 5.07 (d, J = 1.4 Hz, 3H), 4.54–4.47 (m, 3H), 4.16–4.10 (m, 1H), 3.80 (t, J = 5.2 Hz, 2H), 3.62–3.43 (m, 21H), 3.17–3.05 (m, 2H), 3.01–2.85 (m, 4H), 2.72 (d, J = 38.3 Hz, 2H), 2.63–2.52 (m, 2H), 2.40–2.29 (m, 1H), 2.08–2.00 (m, 2H), 1.89 (dd, J = 16.2, 11.8 Hz, 2H), 1.77 (d, J = 13.8 Hz, 2H), 1.63–1.46 (m, 6H), 1.43–1.22 (m, 6H). HRMS (ESI): m / z calcd. For C 53 H 71 N 10 O 13 [M + H] + 1055.5202, found 1055.5202.

[0082] Example 2

[0083] Synthesis of LST-4 NPs:

[0084] Dissolve the LST-4 prepared in Example 1 in acetonitrile with a concentration of 2 mg / mL as the stock solution. Then, slowly inject the stock solution (500 μL) into ultrapure water (5 mL) using a pipette for 0.5 h and sonicate for 2 h. During sonication, remove acetonitrile at a constant rate with nitrogen (the preparation process is as Figure 14 shown).

[0085] Performance test:

[0086] I. Photophysical property test of LST-4 and LST-4 NPs

[0087] 1. Using methanol as the solvent, prepare 6 test concentrations (10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) of the solution of LST-4 prepared in Example 1 by the stepwise dilution method. Using methanol as the blank reference, measure the ultraviolet-visible absorption spectrum of LST-4 in methanol with a UV-2450 (as Figure 1 ). It can be seen that LST-4 has a special absorption peak, and the methanol solutions of LST-4 with different concentrations have a good linear relationship and fitting degree at the maximum absorption peak, reflecting the good concentration dependence of LST-4 (as Figure 2 ).

[0088] 2. Using methanol as the solvent, prepare 6 test concentrations (2.5 μM, 2.0 μM, 1.5 μM, 1.0 μM, 0.5 μM, and 0.25 μM) of the solution of LST-4 prepared in Example 1 by the stepwise dilution method. Using methanol as the blank solvent, measure the fluorescence emission spectrum of LST-4 in methanol with a fluorescence spectrophotometer (as Figure 3 ). It can be seen that LST-4 has a certain fluorescence effect, and there is a certain linear relationship between the fluorescence intensity and the concentration (as Figure 4 ), indicating that there is a concentration dependence between the fluorescence intensity and the concentration of LST-4.

[0089] 3. Using water as the solvent, prepare 5 test concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) of the solution of LST-4 NPs prepared in Example 2 by the stepwise dilution method. Using water as the blank solvent, measure the visual fluorescence imaging of LST-4 NPs with a small animal fluorescence in vivo imager (as Figure 5 ). It can be seen that LST-4 NPs also have a certain fluorescence signal. Furthermore, through the semi-quantitative analysis of the instrument, it shows that there is also a dependence between the fluorescence intensity and the concentration of LST-4 NPs (as Figure 6 ).

[0090] II. Characterization of LST-4 NPs:

[0091] 1. The morphology and size of the synthesized LST-4 NPs were analyzed and characterized by transmission electron microscopy. As Figure 7 shown, it can be seen that they have a spherical nanostructure and are evenly dispersed.

[0092] 2. Figure 8 For the particle size analysis of LST-4 NPs, the particle size of LST-4 NPs is about 146 nm, reflecting a good EPR effect.

[0093] 3. Figure 9 For the potential analysis of LST-4 NPs, it can be seen from the potential analysis results that the potential is negative and the absolute value is greater than 40, indicating that LST-4 NPs have good stability.

[0094] III. In vitro inhibitory effect of LST-4 monomer and LST-4 NPs on HepG 2 cells

[0095] 1. The cytotoxicity was detected by the CCK-8 method. HepG 2 cells were seeded in 96-well plates in groups of 6 replicate wells, cultured overnight with DMEM medium, and then LST-4 NPs were added and incubated overnight. The IC 50 of LST-4 NPs was measured to be 60.58 μg / mL. The IC 50 of LST-4 was measured to be 309.0 μg / mL by the same method. It shows that LST-4 and LST-4 NPs have cytotoxic effects on HepG 2 cells, and the nanoparticle LST-4 NPs can significantly enhance the toxic effect on HepG 2 (as Figure 10 , Figure 11 ).

[0096] 2. Cellular uptake: The fluorescence emissions of LST-4 NPs and Hoechst 33342 (commercial nuclear dye) are 525 nm and 455 nm respectively. Taking the cell nucleus as a reference, the uptake of LST-4 NPs by HepG 2 cells at different incubation times with LST-4 NPs was analyzed (as Figure 12 ). It can be seen from Figure 12 that at 0.5 h, weak green fluorescence of LST-4 NPs appears in the cells, indicating that LST-4 NPs have entered the cells at 0.5 h; as the incubation time prolongs, the green fluorescence gradually increases, and obvious punctate fluorescence (suspected intracellular lysosomes) can be found at 2 h; at 4 h, obvious green fluorescence appears in the cells, indicating that LST-4 NPs have fully entered the cells at this time.

[0097] 3. Subcellular localization: HepG 2 (1×10 4)Cells were seeded in confocal dishes and incubated with LST-4 NPs (20 μg / mL) for 4 h. After incubation, the cells were stained with a combination of three dyes, LysoTracker Red, MitoTracker DeepRed, and Hoechst 33342 for 15 min. After washing twice with PBS and once with blank medium, cell fluorescence imaging was performed to remove excess dyes and nanoparticles. Fluorescence imaging of the cells was performed using a 63× oil immersion objective of a Zeiss Axio Observer7 inverted fluorescence microscope, and analysis was performed using software ZEN 2012 (Carl Zeiss) (the results are shown in Figure 13 ). The fluorescence emission wavelengths of Hoechst 33342, LysoTracker Red, and MitoTracker Deep Red were 455 nm, 512 nm, and 589 nm, respectively. As can be seen from Figure 13 , LST-4 NPs can not only enter the cells smoothly, but also the nanoparticles aggregate in lysosomes.

[0098] In summary, a new matrine derivative (LST-4) was synthesized based on the structural modification of matrine in the present invention. On this basis, nanoparticles (LST-4 NPs) were further self-assembled. LST-4 has new fluorescence properties, with its maximum ultraviolet absorption wavelength being 409 nm and its fluorescence excitation / emission wavelengths being 416 nm / 503 nm. The cytotoxic effects of LST-4 and LST-4 NPs on HepG 2 cells were measured, and it was found that LST-4 NPs can significantly enhance the toxicity to HepG 2 compared with LST-4. In addition, cell uptake experiments and subcellular co-localization experiments of LST-4 NPs were carried out, and it was found that LST-4 NPs can be taken up by cells within 4 h and can aggregate in lysosomes, indicating that LST-4 NPs have good enhanced permeability and retention effects (EPR). Accordingly, LST-4 NPs are expected to have good application value in the integrated diagnosis and treatment of tumors.

[0099] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A matrine derivative, characterized in that, The structural formula of the matrine derivative is as follows:

2. A method for preparing the matrine derivative according to claim 1, characterized in that, It includes the following steps: 1) Dissolve pentaethylene glycol in dichloromethane, then successively add p-toluenesulfonyl chloride and potassium hydroxide powder. After the reaction ends, extract and remove impurities to obtain Compound 1; 2) Add sodium azide to Compound 1 and carry out an oil bath reaction to obtain Compound 2; 3) Mix Compound 2 with triphenylphosphine, react at room temperature, adjust the pH to 14 and then extract to obtain Compound 3; 4) Add thalidomide, N,N-diisopropylethylamine and N,N-dimethylformamide to Compound 3 and carry out an oil bath reaction to obtain Compound 4; 5) Using matrine as a raw material, lithium diisopropylamide as a base, add diphenyldisulfide to carry out a nucleophilic substitution reaction to obtain 14-phenylthio matrine; then mix 14-phenylthio matrine with o-iodoxybenzoic acid for reaction to obtain 14-phenylsulfinyl matrine, and eliminate benzenesulfinic acid under the action of potassium carbonate to obtain Compound 5; Dissolve Compound 5 and glycine in water, heat and react, and recrystallize to obtain Compound 6; Mix methyl Boc-L-tyrosinate, N,N-dimethylformamide solution and potassium carbonate, stir at room temperature, add propargyl bromide, and react at room temperature. After the reaction ends, carry out extraction to obtain Compound 7; Place Compound 7 in a hydrochloric acid ethyl acetate solution, stir at room temperature overnight, remove the solvent after the reaction is completed to obtain Compound 8; Dissolve Compound 6 and Compound 8 in anhydrous dichloromethane, successively add EDCI, HOBt and DIPEA, carry out extraction and impurity removal after the reaction is completed at room temperature to obtain Compound 9; 6) Dissolve Compound 4 and Compound 9 in a tetrahydrofuran solution, stir at room temperature to obtain a reaction solution; Dissolve copper sulfate and sodium ascorbate in water, shake well, and the color of the solution turns brown; Drop the brown aqueous solution into the reaction solution, stir at room temperature to obtain the matrine derivative LST-4; The structural formula of Compound 1 is as follows: The structural formula of Compound 2 is as follows: The structural formula of Compound 3 is as follows: The structural formula of Compound 4 is as follows: The structural formula of Compound 5 is as follows: The structural formula of Compound 6 is as follows: The structural formula of Compound 7 is as follows: The structural formula of Compound 8 is as follows: The structural formula of Compound 9 is as follows:

3. A matrine derivative nanoparticle, characterized in that, Self-assemble the matrine derivative described in Claim 1 to form spherical nano-molecules, namely matrine derivative nanoparticles; Specifically, it includes the following steps: Dissolve the matrine derivative in acetonitrile, inject the obtained solution into ultrapure water, and perform ultrasonic treatment under a nitrogen atmosphere to obtain matrine derivative nanoparticles; The structural formula of the matrine derivative is as follows:

4. A method for preparing matrine derivative nanoparticles, characterized in that, It includes the following steps: Dissolve the matrine derivative in acetonitrile, inject the obtained solution into ultrapure water, and perform ultrasonic treatment under a nitrogen atmosphere to obtain matrine derivative nanoparticles; The structural formula of the matrine derivative is as follows:

5. Use of the matrine derivative nanoparticles as described in Claim 3 in the preparation of anti-tumor drugs.

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