A dandelion flavonoid derivative and its preparation method and application

Abstract

The present invention discloses a dandelion flavonoid derivative, its preparation method, and application. The dandelion flavonoid derivative has the following molecular structure: The dandelion flavonoid derivative provided by the present invention has anti-inflammatory and lung nodule-removing effects, is simple to prepare, and has good biocompatibility, making it a promising new drug for eliminating lung nodules.

Description

Technical Field

[0001] The present invention relates to a derivative, in particular to a dandelion flavonoid derivative and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry. Background Art

[0002] Pulmonary nodules are round or irregular lesions within the lungs with a diameter ≤3 cm. Radiologically, they present as increased density shadows with well-defined or ill-defined lesions, without atelectasis, hilar lymphadenopathy, or pleural effusion. Pulmonary nodules are categorized as solitary or multiple based on their number. Based on their density, they are divided into solid and subsolid nodules. Subsolid nodules are further divided into part-solid and non-solid nodules based on whether they contain solid components. Most nodules are benign, but a small number are malignant. Discrimination between benign and malignant nodules can generally be made based on morphological features combined with dynamic changes. Benign nodules require regular follow-up, while malignant nodules often require early surgical resection.

[0003] The incidence of pulmonary nodules has been increasing in recent years, likely due to factors such as the increasing number of smokers, environmental changes, and the widespread use of low-dose CT (LDCT) screening. Risk factors for pulmonary nodules include genetic risk, environmental factors, occupational factors, dietary habits, infections, and other diseases. Therefore, the development of a novel biocompatible compound that can eliminate pulmonary nodules is of great application value. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a dandelion flavonoid derivative and a preparation method and application thereof.

[0005] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0006] The present invention first proposes a dandelion flavonoid derivative having the following molecular structure expression:

[0007]

[0008] Dandelion flavonoids (3-hydroxyflavone), a bioactive component found in dandelions, possess antioxidant, anti-inflammatory, and anti-tumor properties. Theanine, on the other hand, has anti-fatigue, antihypertensive, and nervous tension-relieving properties. This study combines the two structures through a chemical reaction, unexpectedly discovering that the resulting new substance has the potential to eliminate lung nodules and exhibits excellent biocompatibility.

[0009] Secondly, the present invention also provides a method for preparing a dandelion flavonoid derivative, comprising the following steps:

[0010]

[0011] 1) After theanine and di-tert-butyl dicarbonate are mixed and dissolved, potassium bicarbonate is added and reacted at room temperature to generate compound 1;

[0012] 2) adding compound 1, dandelion flavonoids and 4-dimethylaminopyridine to a solvent to react and generate compound 2;

[0013] 3) After dissolving compound 2, dilute hydrochloric acid was added and stirred at room temperature for 5-24 hours to generate compound 3, i.e., the dandelion flavonoid derivative.

[0014] As a preferred embodiment of the preparation method of the present invention, in step 1), the molar ratio of theanine to di-tert-butyl dicarbonate is 1:(1-1.5), for example, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.; and / or,

[0015] In step 1), the amount of potassium bicarbonate used is 2-5 times the molar amount of theanine, for example, 2.2 times, 2.5 times, 2.8 times, 3 times, 3.5 times, 4 times, 4.5 times, etc.

[0016] As a preferred embodiment of the preparation method of the present invention, in step 1), the reaction solvent is a mixed solvent of diethyl ether and water, preferably with a volume ratio of 1:(0.8-1.2), for example, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, etc.

[0017] Preferably, the reaction time in step 1) is 5-24 h, for example, 6 h, 10 h, 12 h, 15 h, 18 h, 20 h, etc.

[0018] As a preferred embodiment of the preparation method of the present invention, in step 2), the molar ratio of dandelion flavonoids, compound 1 and 4-dimethylaminopyridine is 1:(1-1.2):(0.05-0.2), for example, 1:1:0.1, 1:1.2:0.08, 1:1.15:0.05, 1:1.1:0.2, 1:1.05:0.08, etc.

[0019] As a preferred embodiment of the preparation method of the present invention, in step 2), the reaction solvent is one or more of dichloromethane, chloroform, methanol, ethanol, acetonitrile, tetrahydrofuran, and ethyl acetate;

[0020] Preferably, the reaction conditions in step 2) are reflux reaction for 10-24 h, for example, 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, etc.

[0021] As a preferred embodiment of the preparation method of the present invention, after the reaction in step 2) is completed, dilute hydrochloric acid is added to extract the reaction solution, and the product is purified from the organic phase.

[0022] As a preferred embodiment of the preparation method of the present invention, in step 3), the concentration of dilute hydrochloric acid is 0.1-2 mol / L; and / or, the amount of dilute hydrochloric acid added is 2-5 times that of compound 2 on a molar basis;

[0023] Preferably, the solvent in step 3) is one or more of dichloromethane, chloroform, methanol, ethanol, acetonitrile, tetrahydrofuran, and ethyl acetate.

[0024] As a preferred embodiment of the preparation method of the present invention, after the reaction in step 3) is completed, the reaction solution is adjusted to a pH of 6-7 with a sodium bicarbonate aqueous solution, and the product is purified from the organic phase after extraction with an organic solvent.

[0025] The present invention does not impose any limitation on the specific purification method of the product in steps 1) to 3), which can be extraction, adsorption, recrystallization, column chromatography, or a combination of at least two thereof conventionally used in the art.

[0026] For extraction, the more suitable extraction system is dichloromethane and water. In order to improve the purity and yield of the extract, the product can be recovered through multiple extractions; after extraction, conventional vacuum distillation, crystallization / column chromatography methods can be used for further purification.

[0027] Finally, the present invention also proposes the use of the dandelion flavonoid derivative as described above or the dandelion flavonoid derivative prepared by the method described above in a drug or preparation for eliminating lung nodules.

[0028] The dandelion flavonoid derivative provided by the present invention has the effect of eliminating lung nodules, and has a simple preparation method and good biocompatibility, and is expected to become a new type of drug for treating lung nodules. DETAILED DESCRIPTION

[0029] The present invention will be further described below through specific examples. The examples of the present invention are only for illustration of the present invention and do not limit the scope of the present invention.

[0030] Unless otherwise specified, the main raw materials and reagents in the examples of the present invention are all commercially available products.

[0031] The following Examples 1-3 all prepared dandelion flavonoid derivatives through the following reaction routes:

[0032]

[0033] [Example 1]

[0034] A method for preparing a dandelion flavonoid derivative comprises the following steps:

[0035] 1) Theanine (1 mmol) and di-tert-butyl dicarbonate (1 mmol) were weighed and added to diethyl ether (5 mL) and water (5 mL). Potassium bicarbonate (4 mmol) was then added and reacted at room temperature for 6 h. After the reaction, dichloromethane was added for extraction. The organic phase was dried and then chromatographed on a silica gel column (dichloromethane:methanol, volume ratio = 100:1) to obtain Compound 1.

[0036] 1 H NMR(500MHz,Chloroform-d)δ12.62(s,1H),6.82(d,J=8.2Hz,1H),6.21(t,J= 4.1Hz,1H),4.16(dt,J=8.1,6.5Hz,1H),3.20(qd,J=6.4,4.1Hz,2H),2.34(dt, J=16.3,8.6Hz,1H),2.21(dt,J=16.3,8.8Hz,1H),2.03(dtd,J=15.2,8.7,6.5H z,1H),1.77(dtd,J=15.1,8.7,6.5Hz,1H),1.42(s,9H),1.14(t,J=6.4Hz,3H).

[0037] 2) Compound 1 (1 mmol), dandelion flavonoids (1 mmol), and 4-dimethylaminopyridine (0.1 mmol) were added to dichloromethane (10 mL) and refluxed for 12 h. After the reaction, the reaction mixture was extracted with 0.15 mol / L dilute hydrochloric acid. The organic phase was separated, dried over anhydrous sodium sulfate, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate, volume ratio = 10:1) to obtain compound 2.

[0038] 1H NMR(500MHz,Chloroform-d)δ7.90(dd,J=8.1,1.5Hz,1H),7.42-7.36(m,2H),7.39-7.33(m,3H),7.36-7.27(m ,1H),7.26(dd,J=7.8,1.3Hz,1H),7.13(dd,J=8.2,1.2Hz,1H),6.21(t,J=4.1Hz,1H),5.79-5.64(m,3H),4.35 (dt,J=8.2,6.7Hz,1H),3.20(qd,J=6.4,4.1Hz,2H),2.37(dt,J=16.3,8.5Hz,1H),2.24(dt,J=16.3,8.6Hz,1H ), 2.00(dtd,J=15.0,8.6,6.6Hz,1H),1.75(dtd,J=15.1,8.6,6.6Hz,1H),1.42(s,9H),1.14(t,J=6.4Hz,3H).

[0039] 3) Compound 2 (1 mmol) was added to dichloromethane (10 mL), followed by the addition of 0.15 mol / L dilute hydrochloric acid, and the reaction was stirred at room temperature for 8 h. The amount of dilute hydrochloric acid added was twice the molar amount of compound 2. After the reaction, the reaction mixture was adjusted to pH 7 with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, and then spin-dried to obtain compound 3, a dandelion flavonoid derivative.

[0040] 1 H NMR(500MHz,Chloroform-d)δ7.90(dd,J=8.0,1.5Hz,1H),7.43-7.38(m,1H),7.38(q,J=1.3Hz,1H),7.37-7.34(m,3 H),7.33-7.29(m,1H),7.27(td,J=7.8,1.2Hz,1H),7.13(dd,J=8.3,1.2Hz,1H),6.23(t,J=4.1Hz,1H),5.71(d,J=7. 3Hz,1H),5.66(d,J=7.3Hz,1H),5.47(d,J=6.6Hz,2H),3.69(p,J=6.3Hz,1H),3.20(qd,J=6.4,4.1Hz,2H),2.47-2.3 8(m,1H),2.29(dt,J=16.1,8.4Hz,1H),2.18-1.96(m,1H),1.81(dtd,J=15.4,8.3,6.0Hz,1H),1.14(t,J=6.4Hz,3H).

[0041] [Example 2]

[0042] A method for preparing a dandelion flavonoid derivative comprises the following steps:

[0043] 1) Theanine (1 mmol) and di-tert-butyl dicarbonate (1.5 mmol) were weighed and added to diethyl ether (5 mL) and water (4 mL). Potassium bicarbonate (2 mmol) was then added and allowed to react at room temperature for 10 h. After the reaction, dichloromethane was added for extraction. The organic phase was dried and then chromatographed on a silica gel column (dichloromethane:methanol, volume ratio = 100:1) to obtain Compound 1.

[0044] 2) Compound 1 (0.2 mmol), dandelion flavonoids (1 mmol), and 4-dimethylaminopyridine (0.05 mmol) were added to dichloromethane (10 mL) and refluxed for 12 h. After the reaction, the reaction mixture was extracted with 0.15 mol / L dilute hydrochloric acid. The organic phase was separated, dried over anhydrous sodium sulfate, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate, volume ratio = 10:1) to obtain compound 2.

[0045] 3) Compound 2 (1 mmol) was added to dichloromethane (10 mL), followed by the addition of 1 mol / L dilute hydrochloric acid, and the reaction was stirred at room temperature for 8 h. The amount of dilute hydrochloric acid added was 5 times the molar amount of compound 2. After the reaction, the reaction solution was adjusted to pH 6 with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, and then spin-dried to obtain compound 3, a dandelion flavonoid derivative.

[0046] [Example 3]

[0047] A method for preparing a dandelion flavonoid derivative comprises the following steps:

[0048] 1) Theanine (1 mmol) and di-tert-butyl dicarbonate (1.2 mmol) were weighed and added to ether (5 mL) and water (6 mL). Potassium bicarbonate (5 mmol) was then added and allowed to react at room temperature for 24 h. After completion of the reaction, dichloromethane was added for extraction. The organic phase was dried and then chromatographed on a silica gel column (dichloromethane:methanol, volume ratio = 100:1) to obtain Compound 1.

[0049] 2) Compound 1 (1.1 mmol), dandelion flavonoids (1 mmol), and 4-dimethylaminopyridine (0.2 mmol) were added to dichloromethane (10 mL) and refluxed for 24 h. After the reaction, the reaction mixture was extracted with 0.15 mol / L dilute hydrochloric acid. The organic phase was separated, dried over anhydrous sodium sulfate, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate, volume ratio = 10:1) to obtain compound 2.

[0050] 3) Compound 2 (1 mmol) was added to dichloromethane (10 mL), followed by the addition of 1.5 mol / L dilute hydrochloric acid, and the reaction was stirred at room temperature for 8 h. The amount of dilute hydrochloric acid added was 4 times the molar amount of compound 2. After the reaction, the reaction mixture was adjusted to pH 7 with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, and then spin-dried to obtain compound 3, a dandelion flavonoid derivative.

[0051] Application Examples

[0052] 1. Cell culture and treatment

[0053] Mouse Lewis lung cancer cells were cultured in complete medium containing 10% fetal bovine serum and high-glucose DMEM in a 37°C, 5% CO2 incubator. The culture medium was replaced every 2 days, and the cells were digested and passaged with 0.25% trypsin every 3 days. When the cell confluence reached 90%, the cells were collected, centrifuged, and the supernatant was removed. PBS was added and pipetted to resuspend the cells in PBS. The cell viability was determined to be greater than 95% by trypan blue staining. The cells were then counted and the cell concentration was adjusted to 4×10 7 The cell suspension at 100 μg / mL was prepared for use.

[0054] 2. Preparation of Matrigel before injection

[0055] Matrigel was stored at -20℃ for long term. When in use, it was placed in an ice bath and thawed overnight in a 4℃ refrigerator. The melted Matrigel was mixed with 4×10 7 The cell suspension with a concentration of 100 cells / mL was mixed at a ratio of 1:1 and this operation was performed in an ice bath.

[0056] 3. Methods for establishing an animal model of pulmonary nodules

[0057] Anesthesia: Mice were anesthetized with 10 mg / mL chloral hydrate solution at a dose of 0.05 mL / 10 g body weight.

[0058] Inoculation: After anesthetizing mice, place them in the right lateral position on the operating table. Trim the hair under their arms and disinfect them with ethanol. Make a 5 mm incision approximately 1.5 cm above the costal arch on the left anterior axillary line. Separate the skin and subcutaneous tissue, and expose the chest wall until the lung lobes can be seen moving up and down with breathing. Use a microinjector to place 50 μL of the cell suspension mixed with Matrigel at room temperature for 2 minutes and then inject it into the left lung to a depth of approximately 3 mm. Pause the injection for a few seconds, remove the needle, and suture the incision.

[0059] 4. In vivo experiments

[0060] (1) Animal grouping

[0061] Fifty male mice were randomly divided into five groups, 10 mice in each group: normal saline group, low-dose experimental group, medium-dose experimental group, high-dose experimental group, and raw material control group.

[0062] (2) Reagent preparation

[0063] Normal saline group: 0.9% sodium chloride aqueous solution;

[0064] Low-dose experimental group: The dandelion flavonoid derivative prepared in Example 1 was dissolved in 0.9% sodium chloride aqueous solution to prepare an experimental drug with a concentration of 0.5 mg / mL;

[0065] Medium-dose experimental group: The dandelion flavonoid derivative prepared in Example 1 was dissolved in 0.9% sodium chloride aqueous solution to prepare an experimental drug with a concentration of 1.0 mg / mL;

[0066] High-dose experimental group: The dandelion flavonoid derivative prepared in Example 1 was dissolved in 0.9% sodium chloride aqueous solution to prepare an experimental drug with a concentration of 2.0 mg / mL;

[0067] Raw material control group: Dandelion flavonoids and theanine were dissolved in 0.9% sodium chloride aqueous solution at a mass ratio of 1:1 to prepare an experimental drug with a concentration of 1.0 mg / mL.

[0068] (3) Drug administration experiment

[0069] After the establishment of the animal model of pulmonary nodules, the mice were first raised normally for 6 weeks, and then each group of mice was intragastrically administered with 0.1 mL / 10 g of the drug per day according to their body weight for 2 consecutive months.

[0070] 5. Pathological testing

[0071] After the mice were killed instantly, the lungs of the mice were collected for pathological examination. The pathological sections were prepared according to the conventional HE staining method: xylene I for 10 minutes, xylene II for 20 minutes, 100% ethanol for 5 minutes, 95% and 85% ethanol for 1 minute each, rinsed with tap water for a while, stained with hematoxylin for 10 minutes, rinsed with tap water for 15 minutes, stained with 0.5% eosin for 1 minute, rinsed with tap water, 95% ethanol for a while, 100% ethanol I and II for 10 minutes each, xylene I and II for 10 minutes each, sealed with neutral resin glue, and the mouse nodules were observed under a microscope.

[0072] 6. Experimental Results

[0073] The average diameter of the lung nodules in each group of mice was statistically analyzed after the experiment. The results are shown in Table 1:

[0074] Table 1. Experimental results

[0075] Group Average diameter (mm) Normal saline group 2.1 Low-dose experimental group 1.7 Medium dose experimental group 1.4 High-dose experimental group 1.1 Raw material control group 1.9

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be considered within the scope of protection of the present invention.

Claims

1. A dandelion flavonoid derivative, characterized in that: It has the following molecular structure expression: 。 2. The method for preparing the dandelion flavonoid derivative according to claim 1, characterized in that: The following steps are involved: ； 1) After theanine and di-tert-butyl dicarbonate are mixed and dissolved, potassium bicarbonate is added and reacted at room temperature to produce compound 1; 2) Compound 1, dandelion flavonoids, and 4-dimethylaminopyridine are added to a solvent to react to generate compound 2; 3) After dissolving compound 2, dilute hydrochloric acid was added and stirred at room temperature for 5-24 hours to generate compound 3, i.e., the dandelion flavonoid derivative.

3. The method for preparing dandelion flavonoid derivatives according to claim 2, characterized in that: In step 1), the molar ratio of theanine to di-tert-butyl dicarbonate is 1:(1-1.5); and / or, In step 1), the amount of potassium bicarbonate used is 2-5 times the molar amount of theanine.

4. The method for preparing the dandelion flavonoid derivative according to claim 2 or 3, characterized in that: In step 1), the reaction solvent is a mixed solvent of diethyl ether and water.

5. The method for preparing dandelion flavonoid derivatives according to claim 4, characterized in that: Step 1) In the reaction solvent, the volume ratio of ether to water is 1:(0.8-1.2).

6. The method for preparing the dandelion flavonoid derivative according to any one of claims 2 to 3, characterized in that: In step 2), the molar ratio of dandelion flavonoids, compound 1 and 4-dimethylaminopyridine is 1:(1.1-1.2):(0.03-0.08).

7. The method for preparing dandelion flavonoid derivatives according to claim 5, characterized in that: In step 2), the reaction solvent is one or more of dichloromethane, chloroform, methanol, ethanol, acetonitrile, tetrahydrofuran, and ethyl acetate.

8. The method for preparing dandelion flavonoid derivatives according to claim 7, characterized in that: The reaction conditions in step 2) are reflux reaction for 10-24 hours.

9. The method for preparing a dandelion flavonoid derivative according to any one of claims 2 to 3, characterized in that: After the reaction in step 2) is completed, dilute hydrochloric acid is added to extract the reaction solution, and the product is purified from the organic phase.

10. The method for preparing a dandelion flavonoid derivative according to any one of claims 2 to 3, characterized in that: In step 3), the concentration of dilute hydrochloric acid is 0.1-2 mol / L; and / or the amount of dilute hydrochloric acid added is 2-5 times that of compound 2 in terms of molar amount.

11. The method for preparing dandelion flavonoid derivatives according to claim 10, characterized in that: The solvent in step 3) is one or more of dichloromethane, chloroform, methanol, ethanol, acetonitrile, tetrahydrofuran, and ethyl acetate.

12. The method for preparing a dandelion flavonoid derivative according to any one of claims 2 to 3, characterized in that: After the reaction in step 3) is completed, the reaction solution is adjusted to a pH of 6-7 with a sodium bicarbonate aqueous solution, and the product is purified from the organic phase after extraction with an organic solvent.

13. Use of the dandelion flavonoid derivative according to claim 1 or the dandelion flavonoid derivative prepared by the method according to any one of claims 2 to 12 in the preparation of a drug or preparation for eliminating lung nodules.

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