Berbamine derivatives, preparation method and application thereof

Abstract

This invention discloses a berberine derivative, its preparation method, and its applications, belonging to the field of biomedical technology. The berberine derivative of this invention is a 14-position derivative of berberine. Using berberine as a lead compound, a stepwise synthetic route involving hydroxyl protection, nitration, deprotection, reduction, and substitution is employed to introduce structural units such as amide bonds, sulfonamide bonds, urea bonds, and thiourea bonds at the 14-position, yielding a series of novel derivatives. The preparation method of this invention has a clear synthetic route, high intermediate yields, and is suitable for the synthesis and preparation of target compounds. Some of the berberine derivatives of this invention exhibit good inhibitory activity against MDA-MB-231, 4T1, MDA-MB-468, and MCF-7 breast cancer cell lines, showing promising application potential in the development of anti-breast cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to berberine derivatives, their preparation methods and applications, and especially to the application of berberine derivatives in the preparation of anti-breast cancer drugs. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. Current treatments for breast cancer mainly involve chemotherapy and surgical resection. Chemotherapy primarily uses either the original drug or a targeted drug. However, the original drug has weak antitumor activity, limited inhibitory effect on breast cancer cells, low bioavailability, and significant toxicity, thus limiting its clinical application. Furthermore, the structural modification strategies for the original drug are limited, mostly confined to simple hydroxyl substitution, resulting in a limited range of modifications and a lack of systematic optimization. Summary of the Invention

[0003] This invention proposes a berberine derivative, its preparation method, and its application. By performing targeted structural modification on berberine, it exhibits potent inhibitory activity against breast cancer, while also possessing good safety and drug-like properties.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: The first objective of this invention is to provide a berberine derivative comprising the following general structural formula: ,or ,or ,or .

[0005] Furthermore, in general formula 1, R1 is a group corresponding to an acyl chloride reagent, selected from: , , , , , , , , , , and , The R2 is selected from hydrogen atoms, , , and .

[0006] Furthermore, in general formula 2, R3 is selected from: , , , , , , , , , , , , , and The R4 is selected from hydrogen atoms, , , , and .

[0007] Furthermore, in general formula 3, R5 is selected from: , , , , , and The R6 is selected from hydrogen atoms and .

[0008] Furthermore, in general formula 4, R7 is selected from: , , , , , , , , , and .

[0009] The second objective of this invention is to provide a method for preparing berberine derivatives, comprising the following steps: (1) Weigh berberine (Ber) into a reaction vessel, add dry dichloromethane to dissolve it, and then add triethylamine to form reaction system 1; slowly add acetyl chloride at 0–5℃ for 30–40 min, wash with saturated sodium bicarbonate and extract with dichloromethane 2–3 times, combine the organic phases, dry and filter, and concentrate under reduced pressure to obtain intermediate 1; under argon protection, dissolve intermediate 1 with dry dichloromethane to obtain reaction system 2, add nitric anhydride at 0–5℃ to react, and then wash with saturated sodium bicarbonate and extract with dichloromethane 2–3 times, combine the organic phases, dry and filter, and concentrate under reduced pressure to obtain intermediate 2; (2) Take intermediate 2 and sodium methoxide and dissolve them in methanol. Reflux the reaction mixture in an oil bath at 40–50 °C. After the reaction mixture is concentrated under reduced pressure, dissolve it in dichloromethane. Wash the mixture with saturated sodium chloride aqueous solution and extract it with dichloromethane 2–3 times. Dry the organic phase, filter it, and concentrate it under reduced pressure to obtain intermediate 3. (3) Intermediate 3 and 5% palladium on carbon were placed in methanol, and hydrazine hydrate was added dropwise and refluxed in an oil bath at 70-80℃. The reaction solution was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, dissolved in dichloromethane, washed with saturated sodium chloride aqueous solution and extracted 2-3 times, the organic phase was dried, filtered and concentrated under reduced pressure to obtain intermediate 4. (4) Intermediate 4 and triethylamine were dissolved in dichloromethane under argon protection. The reaction reagent was added at 0°C and reacted for 30 min. The reaction was then carried out at room temperature for 1–2 h. The reaction solution was poured into ice water, adjusted to alkalinity with saturated NaHCO3, extracted with dichloromethane 2–3 times, washed with saturated NaCl, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain a solid. After purification, berberine derivatives were obtained. The reaction reagents are selected from acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates; The mass ratio of intermediate 4 to the reaction reagent is 1:1-1.2.

[0010] Furthermore, the amount of intermediate 1 and nitroacetic anhydride used in step (1) is: 3 mL of nitroacetic anhydride for every 1 g of intermediate 1.

[0011] Furthermore, in step (2), the mass ratio of intermediate 2 to sodium methoxide is 3-5:1, preferably 4-4.5:1.

[0012] Furthermore, the mass ratio of intermediate 3 to palladium on carbon in step (3) is 5-7:1. Preferably, it is 6-6.2:1.

[0013] A third objective of this invention is to provide the use of berberine derivatives or their salts, or formulations containing berberine derivatives, in the preparation of anti-breast cancer drugs. The breast cancer in question is triple-negative breast cancer.

[0014] The beneficial effects of this invention, which relates to berberine derivatives, their preparation methods, and applications, are as follows: (1) The preparation method of the berberine derivative of the present invention has a clear synthetic route. It adopts a stepwise synthetic route of "hydroxyl protection-nitration-deprotection-reduction-electrophilic substitution". First, the C-12 hydroxyl group is protected to avoid side reactions, ensuring the directionality and specificity of the modification. The yield of intermediates in each step is above 88%, which significantly improves the purity and yield of the target product.

[0015] (2) This invention introduces biocompatible functional groups such as amide bonds, sulfonamide bonds, urea bonds, and thiourea bonds at the 14 position of berberine, which can precisely regulate the lipophilicity, water solubility, and spatial conformation of the molecule, thereby optimizing its pharmacokinetic properties. Furthermore, some derivatives exhibit inhibitory activity against four breast cancer cell lines: MDA-MB-231, 4T1, MDA-MB-468, and MCF-7, demonstrating good safety and drug-likeness. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram and a quantitative diagram of the flow cytometry detection of MDA-MB-231 and 4T1 apoptosis in Example 6; where A is a schematic diagram of MDA-MB-231 and 4T1 apoptosis; B is a quantitative diagram of MDA-MB-231 and 4T1 apoptotic cells. Figure 2 This is an image of a subcutaneous tumor in a nude mouse from Example 7; Figure 3 These are the proton NMR spectra of compounds A-01, A-01-2, A-02-2, and A-02. Figure 4 These are the proton NMR spectra of compounds A-03, A-03-2, A-04, and A-05; Figure 5 These are the proton NMR spectra of compounds A-06, A-07, A-08-2, and A-08. Figure 6 These are the proton NMR spectra of compounds A-09, A-10, A-11, and A-12; Figure 7 These are the proton NMR spectra of compounds A-13, B-01, B-02, and B-03. Figure 8 These are the proton NMR spectra of compounds B-04, B-05, B-06, and B-07. Figure 9 These are the proton NMR spectra of compounds B-07-2, B-08, B-09, and B-12. Figure 10 These are the proton NMR spectra of compounds B-10, B-10-2, B-11, and B-11-2. Figure 11 These are the proton NMR spectra of compounds B-13-2, B-13, B-14-2, and B-14. Figure 12 These are the proton NMR spectra of compounds C-01, C-02, C-03, and C-04; Figure 13 These are the proton NMR spectra of compounds C-05-2, C-05, C-06, and C-07; Figure 14 These are the proton NMR spectra of compounds D-01, D-02, D-03, and D-04. Figure 15 These are the proton NMR spectra of compounds D-05, D-06, D-07, and D-08; Figure 16 These are the proton NMR spectra of compounds D-09, D-10, D-11, and B-15. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0019] Example 1 A method for preparing a berberine derivative includes the following steps: Step (1) Weigh 1.00 g (1.64 mmol) of berberine (Ber) into a 100 ml round-bottom flask and dissolve it in 40 ml of dry dichloromethane (DCM). Then add triethylamine (685.03 ml, 4.93 mmol). Place the reaction system in an ice-water bath and stir for 2-3 min to lower the temperature of the reaction system to 0-5℃. Slowly add acetyl chloride (175.20 ml, 2.46 mmol) and stir in an ice-water bath for 30 min. TLC showed no raw material remaining. Slowly add saturated sodium bicarbonate solution to wash the reaction solution. Extract with DCM 2-3 times, collect and combine the organic phases, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain 1.26 g of pale yellow crude product. The crude product is purified by column chromatography (PE / EA = 2:1, v / v) to obtain 1.02 g of pale yellow pure intermediate 1, with a yield of 95%. (2) Take a 10 mL dry round-bottom reaction flask, add 2.00 mL of acetic anhydride to it under argon protection, place it in an ice-water bath, and wait for the reaction system to cool to 0-5℃. Slowly add 1.40 mL (21.00 mmol, 95%) of concentrated nitric acid to the reaction system and stir rapidly. After stirring in an ice-water bath for 20 min, a pale yellow nitrating reagent, nitric anhydride, is obtained and placed in an ice bath for later use. Intermediate 1 (1.00 g, 1.65 mmol) was weighed and placed in a 50 mL round-bottom flask. Air was removed from the system through three vacuum argon purging cycles. Then, under argon protection, 30.00 mL of dry DCM was added to the flask, and the mixture was stirred until BER-OAC was completely dissolved. The reaction system was then placed in an ice-water bath and stirred for 2-3 min. After the system temperature was lowered to 0-5℃ and stabilized, 3.00 mL of freshly prepared nitroacetic anhydride was slowly added dropwise to the reaction system. After the addition was complete, the reaction was continued under ice-water bath conditions. TLC showed no remaining starting material. The mixture was washed with saturated sodium bicarbonate solution, extracted 2-3 times with DCM, and the organic phases were collected and combined. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 1.58 g of orange crude product. The crude product was purified by column chromatography (PE / EA = 2:1, v / v) to obtain 1.05 g of yellow pure intermediate 2, with a yield of 98%. (3) Weigh intermediate 2 (1.00 g, 1.44 mmol) and sodium methoxide (232.94 mg, 4.31 mmol), place them in a 100 ml round-bottom flask, add 45 ml of methanol, install a reflux condenser, stir under an oil bath, and slowly raise the system temperature from room temperature to 40-50℃, maintaining this temperature. TLC showed no raw material remaining. Concentrate the reaction solution in the reaction flask under reduced pressure, dissolve it with DCM, wash the organic phase with saturated sodium chloride aqueous solution, extract with DCM 2-3 times, collect and combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 1.30 g of yellow crude product. The crude product is purified by column chromatography (PE / EA = 1:1, v / v) to obtain 826.83 mg of pale yellow pure intermediate 3, with a yield of 88%. (4) Weigh intermediate 3 (1.00 g, 1.53 mmol) and 5% palladium on carbon (163.29 mg, 767.21 µmol) into a 50 ml round-bottom flask, add 25 mL of methanol, and then dropwise add hydrazine hydrate (3.00 mL, 49.10 mmol). Install a reflux condenser, stir under an oil bath, and slowly raise the temperature of the system from room temperature to 80 °C for reflux. TLC showed no remaining raw material. A sintered sand funnel was taken, filled with diatomaceous earth, and completely moistened with a small amount of methanol. The reaction system was poured onto the moistened diatomaceous earth and filtered. The diatomaceous earth in the sintered sand funnel was washed with methanol 3-4 times. The filtrate was concentrated under reduced pressure and dissolved in DCM. The organic phase was washed with saturated sodium chloride aqueous solution and extracted with DCM 2-3 times. The organic phases were collected and combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 1.50 g of yellow crude product. The crude product was purified by column chromatography (PE / EA = 2:1, v / v) to obtain 981.00 mg of pale yellow pure intermediate 4, with a yield of 98%. (5) Take 100 mg of dried intermediate 4 and 68 µL of triethylamine in a 10 mL round-bottom flask, add 2 mL of DCM under Ar protection and stir to dissolve the sample. Add acyl chloride reagent at 0 °C and place at room temperature after 30 min. The reaction was detected by TLC and the reaction was complete in 1-2 h. Pour the reaction solution into ice water, adjust the pH to alkaline with saturated NaHCO3 aqueous solution, extract with DCM 3 times, wash with saturated NaCl solution, dry with anhydrous sodium sulfate, filter and combine the organic phases, and recover the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography with DCM:MeOH = 50:1, v / v to obtain the berberine derivative.

[0020] In step (5), different acyl chloride reagents are added, resulting in different products.

[0021] Taking general formula 1 as an example: The specific acyl chloride reagents and their corresponding compound structural formulas are shown in Table 1: Table 1. R1 and R2 groups corresponding to different reagents in Formula 1

[0022] The spectral data for each group are as follows: Intermediate 1: Yellow solid; 95% yield; 1 H NMR (600 MHz, Chloroform-d)) δ 7.5(dd,1H), 7.2 – 7.1 (m, 1H), 6.9 – 7.0(m, 2H), 6.9 – 6.8 (m, 2H), 6.6 (s, 1H),6.4 – 6.5(m, 2H), 6.4 – 6.3 (m, 2H), 5.5 (d, 1H), 4.3 (d, 1H), 3.8 (d, 3H), 3.6 (s, 2H), 3.5 – 3.4 (m, 1H), 3.4 (s, 1H), 3.3 – 3.2 (m, 2H), 3.2 (s, 2H),3.1 (s, 1H), 3.1 – 3.0 (m, 2H), 2.8 – 2.7 (m, 4H), 2.7 (s, 2H), 2.6 (s, 1H), 2.6 (s, 2H), 2.4 (dd, 2H), 2.3 (d, 3H). ESI-MS: m / z 650.77, [M+H] + .

[0023] Intermediate 2: Yellow solid; yield 98%;1 H NMR (600 MHz, Chloroform-d) δ 7.4 (dd,2H), 7.1 (d, 1H), 7.1 – 7.0 (m, 1H), 6.9 (dd, 2H), 6.9 – 6.9 (m, 3H), 6.9(dd, 2H), 6.9 – 6.8 (m, 1H), 6.7 (s, 2H), 6.5 – 6.4 (m, 1H), 6.4 (s, 2H), 6.3– 6.3 (m, 2H), 5.5 (s, 2H), 4.4 – 4.3 (m, 1H), 4.3 (d, 2H), 3.8 (d, 7H), 3.8– 3.8 (m, 2H), 3.7 (s, 5H), 3.6 (d, 1H), 3.5 (s, 2H), 3.3 (d, 3H), 3.3 (s,5H), 3.2 (s, 2H), 3.1 (td, 3H), 3.0 (m, 3H), 2.9 (m, 6H), 2.8 – 2.8 (m, 3H),2.7 (d, 2H), 2.7 (d, 1H), 2.7 (s, 6H), 2.6 (d, 6H), 2.6 (d, 2H), 2.5 (d, 3H),2.4 – 2.4 (m, 2H), 2.3 (d, 7H), 2.2 (s, 1H), 2.2 (s, 1H). ESI-MS: m / z 695.28[M+H] + .

[0024] Intermediate 3: Yellow solid; yield 88%; 1H NMR (600 MHz, Chloroform-d) δ 7.4 (dd,1H), 7.0 – 6.9 (m, 3H), 6.8 (d, 1H), 6.7 (dd, 1H), 6.6 (d, 1H), 6.3 (s, 1H),6.3 (dd, 1H), 5.4 (d, 1H), 5.3 (s, 1H), 4.3 (d, 1H), 4.1 (q, 2H), 3.8 (d,4H), 3.7 (s, 3H), 3.6 – 3.6 (m, 1H), 3.4 (s, 1H), 3.3 (d, 1H), 3.2 (s, 3H),3.2 (s, 1H), 3.1 (d, 1H), 3.1 – 3.1 (m, 1H), 3.0 (m, 1H), 3.0 – 2.9 (m, 2H), 2.8 – 2.7 (m, 4H), 2.6 (s, 3H), 2.6 (s, 1H), 2.6 (s, 3H), 2.5 – 2.5 (m, 1H), 2.5 (s, 1H), 2.4 (dd, 2H), 2.0 (s, 2H). ESI-MS: m / z 653.27 [M+H] + .

[0025] Intermediate 4: Yellow solid; yield 98%; 1 H NMR (600 MHz, Chloroform-d) δ 7.4 (dd,1H), 7.0 – 6.9 (m, 2H), 6.8 (d, 1H), 6.7 (d, 1H), 6.7 (dd, 1H), 6.6 (d, 1H),6.4 (d, 1H), 6.3 (d, 1H), 5.4 (d, 1H), 4.3 – 4.2 (m, 1H), 3.8 (s, 3H), 3.6 (s, 2H), 3.6 (dd, 1H), 3.4 – 3.2 (m, 6H), 3.2 – 3.1 (m, 1H), 3.1 (dd, 1H),3.0 (dd, 1H), 2.9 – 2.6 (m, 5H), 2.6 (s, 2H), 2.5 (d, 3H), 2.4 – 2.3 (m, 4H), 1.9 (s, 1H). ESI-MS: m / z 623.30 [M+H] + .

[0026] A-01: Yellow solid; yield 80%; 1H NMR (600 MHz, Chloroform-d) 7.94 (d, 1H),7.83 (t, 1H), 7.62 (s, 1H), 7.55 (d, 1H), 7.52 – 7.49 (m, 1H), 7.45 (t, 1H),7.29 (q, 1H), 7.02 (s, 1H), 6.93 (m, 1H), 6.87 – 6.78 (m, 1H), 6.65 (d, 1H), 6.41 (d, 1H), 6.36 – 6.32 (m, 1H), 5.37 – 5.26 (m, 1H), 4.52 (d, 1H), 4.40(d, 1H), 3.85(s, 1H), 3.73 (s, 3H), 3.65 (s, 2H), 3.57 (s, 1H), 3.47 – 3.38 (m, 2H), 3.27 (d, 3H), 3.21 – 3.09 (m, 1H), 3.05 (dd, 1H), 2.99 – 2.74 (m,5H), 2.68 (s, 2H), 2.61 (d, 5H), 2.38 (dt, 1H); ESI-MS: m / z 761.29 [M+H] + .

[0027] A-02: Yellow solid; yield 70%; 11H NMR (600 MHz, Methanol-d4) δ 8.0 (dd, 3H), 7.6 (dd, 3H), 7.5 – 7.4 (m, 1H), 7.4 (dd, 1H), 7.0 – 7.0 (m, 2H), 7.0 – 6.9 (m, 3H), 6.8 – 6.8 (m, 2H), 6.8 – 6.7 (m, 2H), 6.6 (d, 2H), 6.4 (dd, 1H), 5.4 (d, 1H), 4.5 (dd, 2H), 3.9 – 3.8 (m, 2H), 3.8 (d, 5H), 3.7 (s, 3H), 3.7 (q, 2H), 3.6 (s, 1H), 3.4 (s, 2H), 3.4 – 3.3 (m, 1H), 3.3 (s, 2H), 3.3 – 3.2 (m, 3H), 3.2 (s, 2H), 3.1 (d, 1H), 3.1 (dd, 2H), 3.0 – 2.9 (m, 7H), 2.8 (d, 5H), 2.7 (s, 2H), 2.7 (s, 2H), 2.6 (s, 2H), 2.5 (d, 1H); ESI-MS: m / z 761.29 [M+H] + .

[0028] A-02-2: Yellow solid; Yield 87%; 1 1H NMR (600 MHz, Chloroform-d) δ 8.1 (dd, 2H), 7.9 (d, 2H), 7.5 (dd, 4H), 7.4 – 7.3 (m, 2H), 7.0 (d, 1H), 6.9 (m, 2H), 6.9 (dd, 1H), 6.7 (s, 1H), 6.4 (d, 1H), 6.3 (dd, 1H), 5.6 (d, 1H), 4.2 (d, 1H), 3.7 (s, 3H), 3.7 (d, 3H), 3.4 (s, 1H), 3.3 (d, 3H), 3.2 – 3.1 (m, 2H), 2.9 – 2.8 (m, 3H), 2.8 – 2.7 (m, 2H), 2.7 (m, 1H), 2.6 (s, 2H), 2.6 (d, 3H), 2.5 (s, 1H), 2.4 (t, 2H), 2.2 – 2.0 (m, 1H). ESI-MS: m / z 899.27 [M+H] + .

[0029] A-03: White solid; yield 83%; 1 H NMR (600 MHz, Chloroform-d) δ 7.84 (d,2H), 7.49 – 7.39 (m, 2H), 7.31 (d, 2H), 7.00 – 6.92 (m, 2H), 6.78 (d, 1H),6.73 (dd, 1H), 6.66 (d, 1H), 6.46 – 6.29 (m, 2H), 5.41 (d, 1H), 4.30 (dd,1H), 3.72 (s, 3H), 3.64 (d, 3H), 3.52 – 3.45 (m, 1H), 3.39 (s, 1H), 3.32 (d,1H), 3.25 (s, 2H), 3.21 – 3.16 (m, 2H), 3.12 (dd, 1H), 3.01 (dd, 1H), 2.92(dd, 2H), 2.83 (m, 2H), 2.78 – 2.74 (m, 2H), 2.71 – 2.66 (m, 1H), 2.64 (s,2H), 2.58 (s, 1H), 2.56 (s, 2H), 2.49 (s, 1H), 2.41 – 2.36 (m, 2H); ESI-MS:m / z 739.35 [M+H] + .

[0030] A-03-2: Yellow solid; yield 82%; 11H NMR (600 MHz, Chloroform-d) δ 8.09 (dd, 2H), 7.84 (d, 2H), 7.46 – 7.35 (m, 2H), 7.34 – 7.27 (m, 4H), 7.01 (d, 1H), 6.94 (m, 2H), 6.87 (dd, 1H), 6.67 (d, 1H), 6.44 – 6.32 (m, 2H), 5.59 (d, 1H), 4.35 – 4.23 (m, 1H), 3.72 (s, 3H), 3.67 (d, 3H), 3.42 (s, 1H), 3.31 – 3.25 (m, 3H), 3.23 – 3.12 (m, 3H), 2.92 – 2.87 (m, 2H), 2.83 – 2.77 (m, 2H), 2.76 – 2.70 (m, 2H), 2.64 (s, 3H), 2.59 (d, 3H), 2.51 (s, 1H), 2.44 (s, 4H), 2.42 (s, 4H), 2.39 (d, 1H); ESI-MS: m / z 858.39 [M+H] + .

[0031] A-04: Beige solid; Yield 82%; 1 1H NMR (600 MHz, Chloroform-d) δ 7.4 (dd, 1H), 7.3 (s, 1H), 7.0 – 6.9 (m, 3H), 6.8 (d, 1H), 6.7 – 6.7 (m, 1H), 6.6 (d, 2H), 6.4 (d, 1H), 6.3 (q, 2H), 5.4 (d, 1H), 4.2 (d, 1H), 3.7 (s, 4H), 3.6 (d, 4H), 3.4 (s, 1H), 3.3 (s, 1H), 3.2 (s, 3H), 3.1 (d, 3H), 2.9 (s, 2H), 2.9 (s, 2H), 2.9 – 2.8 (m, 3H), 2.8 – 2.7 (m, 1H), 2.7 (s, 1H), 2.6 (s, 3H), 2.6 (d, 4H), 2.5 (s, 1H), 2.4 – 2.4 (m, 5H), 1.8 (t, 2H), 1.4 (d, 5H), 1.3 – 1.2 (m, 2H). ESI-MS: m / z 721.37 [M+H] + .

[0032] A-05: Yellow solid; yield 91%; 1 H NMR (600 MHz, Methanol- d4) δ 8.0 (dd,2H), 7.6 – 7.6 (m, 1H), 7.6 – 7.5 (m, 3H), 7.4 (td, 1H), 7.0 (d, 1H), 7.0 –6.9 (m, 2H), 6.9 – 6.8 (m, 1H), 6.8 – 6.7 (m, 1H), 6.6 (d, 1H), 6.6 – 6.4 (m,1H), 5.4 (dd, 1H), 4.6 (dd, 1H), 4.3 (s, 1H), 3.8 – 3.7 (m, 5H), 3.5 – 3.4(m, 3H), 3.4 – 3.3 (m, 1H), 3.3 – 3.3 (m, 2H), 3.3 – 3.1 (m, 3H), 3.0 (dd,2H), 2.9 (d, 2H), 2.9 (d, 2H), 2.8 – 2.8 (m, 1H), 2.7 (d, 4H), 2.5 (dt, 1H);ESI-MS: m / z 727.33 [M+H] + .

[0033] A-06: White solid, yield 83%; 1H NMR (600 MHz, Chloroform-d) δ 9.34 (d, 1H),8.22 (m, 1H), 7.82 – 7.62 (m, 1H), 7.54 – 7.47 (m, 1H), 7.11 (t, 1H), 7.08 –7.00 (m, 2H), 6.91 (t, 1H), 6.87 – 6.78 (m, 2H), 6.64 – 6.53 (m, 1H), 6.44 –6.36 (m, 1H), 6.30 (dd, 1H), 5.25 (s, 1H), 4.70 – 4.51 (m, 1H), 4.05 (d, 3H), 3.96 (d, 1H), 3.85 (q, 1H), 3.71 (d, 3H), 3.65 (s, 2H), 3.49 (s, 1H), 3.40(d, 2H), 3.25 (s, 2H), 3.16 (s, 2H), 3.14 – 3.04 (m, 1H), 3.04 – 2.95 (m,2H), 2.93 – 2.79 (m, 3H), 2.74 (s, 2H), 2.66 (d, 5H), 2.37 (d, 1H); ESI-MS:m / z 757.34 [M+H] + .

[0034] A-07: Yellow solid, yield 82%; 11H NMR (600 MHz, Chloroform-d) δ 7.85 (d, 2H), 7.58 – 7.49 (m, 2H), 7.04 (s, 1H), 6.97 – 6.88 (m, 2H), 6.88 – 6.79 (m, 2H), 6.65 (s, 1H), 6.45 – 6.32 (m, 2H), 5.31 (d, 1H), 4.42 (d, 1H), 3.98 (s, 1H), 3.90 (s, 1H), 3.74 (s, 4H), 3.66 (s, 3H), 3.63 – 3.55 (m, 2H), 3.46 (d, 2H), 3.41 (s, 2H), 3.35 (d, 3H), 3.24 (s, 3H), 3.17 (s, 2H), 3.09 – 3.01 (m, 2H), 2.99 – 2.91 (m, 3H), 2.90 – 2.75 (m, 5H), 2.70 (s, 3H), 2.63 (d, 6H), 2.44 – 2.36 (m, 1H); ESI-MS: m / z 795.25 [M+H] + . A-07-2: Yellow solid, yield 89%; 1 1H NMR (600 MHz, Chloroform-d) δ 8.09 (dd, 2H), 7.84 (d, 2H), 7.46 – 7.35 (m, 2H), 7.34 – 7.27 (m, 4H), 7.01 (d, 1H), 6.94 (m, 2H), 6.87 (dd, 1H), 6.67 (d, 1H), 6.44 – 6.32 (m, 2H), 5.59 (d, 1H), 4.35 – 4.23 (m, 1H), 3.72 (s, 3H), 3.67 (d, 3H), 3.42 (s, 1H), 3.31 – 3.25 (m, 3H), 3.23 – 3.12 (m, 3H), 2.92 – 2.87 (m, 2H), 2.83 – 2.77 (m, 2H), 2.76 – 2.70 (m, 2H), 2.64 (s, 3H), 2.59 (d, 3H), 2.51 (s, 1H), 2.44 (s, 4H), 2.42 (s, 4H), 2.39 (d, 1H); ESI-MS: m / z 969.73 [M+H] + .

[0035] A-08: Yellow solid, yield 85%; 1 H NMR (600 MHz, Chloroform-d) δ 7.94 – 7.82(m, 2H), 7.64 – 7.59 (m, 1H), 7.55 – 7.42 (m, 3H), 7.15 (d, 1H), 6.86 – 6.79(m, 2H), 6.53 (s, 1H), 6.40 (dd, 1H), 6.28 (s, 1H), 5.89 (dd, 1H), 5.25 (d,1H), 4.38 (d, 1H), 3.75 (s, 5H), 3.60 (s, 3H), 3.50 (dd, 2H), 3.37 (s, 1H),3.25 (s, 4H), 2.79 (td, 3H), 2.69 (s, 3H), 2.67 – 2.58 (m, 2H), 2.57 (s, 3H), 2.47 – 2.38 (m, 2H), 2.26 (m, 1H); ESI-MS: m / z 796.74 [M+H] + .

[0036] A-09: White solid, yield 63%; 1 H NMR (600 MHz, Chloroform-d) δ 7.90 (s,2H), 7.64 (s, 1H), 7.53 (s, 2H), 7.03 (s, 1H), 6.92 (s, 2H), 6.83 (s, 2H),6.64 (s, 1H), 6.42 (s, 1H), 6.34 (s, 1H), 5.33 (s, 1H), 4.54 (s, 1H), 4.41(s, 1H), 3.99 (s, 1H), 3.83 (s, 1H), 3.72 (s, 3H), 3.65 (s, 3H), 3.55 (s,1H), 3.47 (s, 1H), 3.41 (s, 1H), 3.31 (s, 2H), 3.25 (s, 2H), 3.18 (s, 1H), 3.11 (s, 1H), 2.99 (s, 3H), 2.85 (s, 5H), 2.68 (s, 3H), 2.62 (s, 4H), 2.53(s, 2H), 2.38 (s, 1H); ESI-MS: m / z 783.39 [M+H] + .

[0037] A-10: Yellow solid, yield 84%; 1 H NMR (600 MHz, Chloroform-d) δ 7.88 (d,1H), 7.59 – 7.51 (m, 1H), 7.09 – 7.01 (m, 1H), 6.93 – 6.83 (m, 3H), 6.80 (d,1H), 6.66 – 6.59 (m, 2H), 6.40 – 6.31 (m, 2H), 5.30 (d, 1H), 4.54 (d, 1H), 4.41 (d, 1H), 4.09 (s, 3H), 3.91 (s, 1H), 3.74 (d, 4H), 3.64 (s, 3H), 3.54(d, 3H), 3.50 (d, 1H), 3.38 (s, 2H), 3.23 (s, 2H), 3.16 (s, 2H), 3.09 (t,1H), 2.93 – 2.75 (m, 7H), 2.68 (s, 3H), 2.65 (d, 4H), 2.61 (d, 3H), 2.43 –2.36 (m, 1H); ESI-MS: m / z 695.32 [M+H] + .

[0038] A-11: Yellow solid, yield 80%; 1 H NMR (600 MHz, Chloroform-d) δ 7.7 – 7.6 (m,2H), 7.5 (s, 1H), 7.4 (t, 1H), 7.1 – 7.0 (m, 1H), 7.0 – 6.9 (m, 1H), 6.9 (dd,1H), 6.8 (d, 1H), 6.6 (d, 2H), 6.4 (d, 1H), 6.4 – 6.3 (m, 2H), 5.2 (d, 1H), 4.6 (dd, 2H), 4.1 (s, 1H), 3.8 (d, 5H), 3.7 (s, 4H), 3.4 (d, 3H), 3.2 (s,4H), 3.1 (d, 5H), 3.0 – 2.9 (m, 7H), 2.9 – 2.8 (m, 3H), 2.7 (dd, 9H), 2.6 (d,5H), 2.4 (dt, 2H), 1.2 (s, 3H); ESI-MS: m / z 741.34 [M+H] +.

[0039] A-12: Yellow solid, yield 64%; 1 H NMR (600 MHz, Chloroform-d) δ 7.8 (d, 2H),7.4 – 7.3 (m, 3H), 7.1 – 7.0 (m, 1H), 6.9 – 6.8 (m, 3H), 6.7 – 6.6 (m, 2H),6.5 (d, 1H), 6.4 – 6.3 (m, 1H), 5.2 (s, 1H), 4.7 (dd, , 1H), 4.3 (s, 1H), 3.9(d, 2H), 3.8 – 3.6 (m, 8H), 3.6 (d, 2H), 3.4 (s, 2H), 3.3 (d, 3H), 3.2 (s,3H), 3.1 (s, 2H), 3.1 – 3.0 (m, 3H), 2.9 (s, 3H), 2.8 (d, 5H), 2.7 (s, 2H), 2.4 (d, 5H); ESI-MS: m / z 741.34 [M+H] + .

[0040] A-13: White solid, yield 70%; 1 H NMR (600 MHz, Chloroform-d) δ 8.22 (d, 1H),7.62 (s, 1H), 7.52 (s, 1H), 7.08 (s, 3H), 6.89 (s, 3H), 6.80 (m, 1H), 6.64(s, 1H), 6.44 (dt, 1H), 6.35 (s, 1H), 5.29 – 5.21 (m, 1H), 4.58 (s, 1H), 4.05(m, 4H), 3.72 (s, 3H), 3.65 (s, 2H), 3.59 (s, 1H), 3.45 (s, 1H), 3.41 (s,2H), 3.26 (s, 2H), 3.17 (d, 2H), 3.03 (s, 2H), 2.94 (d, 4H), 2.82 (s, 1H), 2.73 (s, 2H), 2.69 (s, 3H), 2.66 (d, 2H), 2.39 (s, 1H); ESI-MS: m / z 757.34 [M+H] + .

[0041] Example 2 A method for preparing a berberine derivative includes the following steps: Steps (1)-(4) of Example 2 are the same as steps (1)-(4) of Example 1; (5) Take 100 mg of dried intermediate 4 and 68 µL of triethylamine in a 10 mL round-bottom flask, add 2 mL of DCM under Ar protection and stir to dissolve the sample. Add sulfonyl chloride reagent at 0 °C and place at room temperature after 30 min. The reaction was detected by TLC and was completed in 1-2 h. Pour the reaction solution into ice water, adjust the pH to alkaline with saturated NaHCO3 aqueous solution, extract with DCM 3 times, wash with saturated NaCl solution, dry with anhydrous sodium sulfate, filter and combine the organic phases, and recover the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography with DCM:MeOH = 50:1, v / v to obtain the berberine derivative.

[0042] In step (5), different sulfonyl chloride reagents are added, resulting in different products.

[0043] Take general formula 2 as an example: The specific sulfonyl chloride reagents and their corresponding compound structures are shown in Table 2: Table 2. R3 and R4 groups corresponding to different reagents in Formula 2

[0044] The spectral data for each substance are as follows: B-01: Yellow solid, yield 83%; 1 H NMR (600 MHz, Chloroform-d) δ 8.17 (s,1H), 8.08 (s, 1H), 7.88 (s, 1H), 7.61 (s, 2H), 7.17 (s, 1H), 6.87 (d, 2H),6.54 (s, 1H), 6.45 (s, 1H), 6.34 (s, 1H), 5.85 (s, 1H), 4.42 (s, 1H), 3.76(s, 4H), 3.61 (s, 3H), 3.52 (s, 2H), 3.38 (s, 1H), 3.26 (s, 5H), 4.21 (m,4H), 2.86 – 2.73 (m,3H), 2.72 (s, 3H), 2.61 (s, 4H), 2.44 (s, 2H), 2.28 (s,1H); ESI-MS: m / z 788.29 [M+H] + .

[0045] B-02: Yellow solid, yield 85%; 1 H NMR (600 MHz, Chloroform-d) δ 7.97 (d,2H), 7.73 (d, 2H), 7.61 (d, 1H), 7.15 (d, 1H), 6.85 (dd, 2H), 6.58 – 6.53 (m,2H), 6.30 (s, 1H), 5.67 (dd, 1H), 4.39 (d, 1H), 3.75 (s, 5H), 3.62 (s, 3H), 3.57 (d, 1H), 3.48 (d, 1H), 3.36 (s, 1H), 3.24 (s, 5H), 3.09 (q, 1H), 2.80 (m, 3H), 2.70 (s, 4H), 2.69 – 2.60 (m, 2H), 2.57 (s, 3H), 2.44 (dd, 2H), 2.23(m, 1H); ESI-MS: m / z 788.29 [M+H] + .

[0046] B-03: Yellow solid, yield 72%; 1 H NMR (600 MHz, Chloroform-d) δ 7.8 – 7.7(m, 2H), 7.5 – 7.4 (m, 3H), 7.2 (d, 1H), 6.9 – 6.8 (m, 2H), 6.5 (s, 1H), 6.4(dd, 1H), 6.3 (s, 1H), 5.8 (dd, 1H), 5.3 (d, 1H), 4.3 (s, 1H), 3.8 (s, 3H), 3.7 (s, 1H), 3.6 (s, 3H), 3.4 (t, 2H), 3.3 (s, 3H), 3.2 – 3.1 (m, 2H), 2.8(m, 2H), 2.7 – 2.7 (m, 2H), 2.7 (s, 3H), 2.6 (s, 3H), 2.6 – 2.5 (m, 1H), 2.5– 2.4 (m, 2H), 2.3 – 2.2 (m, 1H), 1.3 (m, 9H) ; ESI-MS: m / z 819.36 [M+H] + .

[0047] B-04: Yellow solid, yield 89%; 1H NMR (600 MHz, Chloroform-d) δ 8.9 (d, 1H),8.8 (dd, 1H), 8.2 (dt, 1H), 7.5 (d, 1H), 7.4 (dd, 1H), 7.2 (d, 1H), 6.9 – 6.8(m, 2H), 6.5 (s, 1H), 6.4 – 6.4 (m, 1H), 6.3 (s, 1H), 5.8 (dd, 1H), 5.3 (d,1H), 4.4 (d, 1H), 3.7 (s, 4H), 3.6 (s, 3H), 3.5 – 3.4 (m, 2H), 3.4 (s, 1H),3.2 (s, 4H), 3.2 (d, 1H), 2.8 – 2.7 (m, 3H), 2.7 (s, 3H), 2.7 – 2.6 (m, 1H), 2.5 – 2.3 (m, 2H), 2.2 (m, 1H); ESI-MS: m / z 764.29 [M+H] + .

[0048] B-05: Yellow solid, yield 82%; 1 H NMR (600 MHz, Chloroform-d) δ 8.63 (d,1H), 8.13 (dd, 1H), 7.62 (d, 1H), 7.41 (d, 1H), 7.22 (d, 1H), 6.92 – 6.84 (m,2H), 6.58 – 6.50 (m, 2H), 6.33 (s, 1H), 5.67 (dd, 1H), 5.25 (d, 1H), 4.40 (d,1H), 3.76 (s, 4H), 3.63 (s, 3H), 3.59 (d, 1H), 3.52 (d, 1H), 3.25 (s, 4H),3.09 (q, 1H), 2.82 (dd, 1H), 2.80 – 2.74 (m, 2H), 2.70 (s, 4H), 2.62 (d, 1H), 2.58 (s, 3H), 2.45 (dd, 2H), 2.26 (t, 1H); ESI-MS: m / z 798.25 [M+H] + .

[0049] B-06: Yellow solid, yield 77%; 1H NMR (600 MHz, Chloroform-d) δ 8.68 (t,1H), 8.44 (m, 1H), 8.28 (dt, 1H), 7.67 (t, 1H), 7.30 (dd, J1H), 7.17 (d, 1H),6.82 (m, 2H), 6.46 (dd, 1H), 6.27 (s, 1H), 5.74 (dd, 1H), 5.31 (d, 2H), 4.12(d, 1H), 3.75 (s, 4H), 3.60 (s, 4H), 3.39 – 3.30 (m, 1H), 3.25 (s, 5H), 3.16– 3.10 (m, 1H), 3.01 (dd, 1H), 2.78 – 2.68 (m, 4H), 2.61 (s, 3H), 2.54 (s,3H), 2.43 – 2.32 (m, 3H), 2.23 (m, 1H); ESI-MS: m / z 1061.10 [M+H] + .

[0050] B-07-2: Yellow solid, 86% yield; ¹H NMR (600 MHz, Chloroform-d) δ 7.95 (s, 2H), 7.78 (s, 1H), 7.69 (s, 1H), 7.37 (qtd, 6H), 7.05 (s, 1H), 6.84 (s, 1H), 6.76 (s, 1H), 6.52 (s, 1H), 6.43 (s, 1H), 6.24 (s, 1H), 6.05 (s, 1H), 4.22 (d, 1H), 3.65 (s, 1H), 3.55 (s, 3H), 3.37 (t, 3H), 3.29 (s, 3H), 3.24 – 3.12 (m, 4H), 3.03 (s, 1H), 2.99 (s, 3H), 2.80 (s, 1H), 2.77 – 2.66 (m, 5H), 2.63(s, 3H), 2.53 (s, 4H), 2.36 (d, 2H), 2.22 (s, 1H); ESI-MS: m / z 1061.10 [M+H] + .

[0051] B-07: Yellow solid, yield 82%; 1H NMR (600 MHz, Chloroform-d) δ 8.00 – 7.92(m, 1H), 7.68 (dd, 1H), 7.48 (d, 1H), 7.40 – 7.34 (m, 2H), 7.06 – 6.98 (m,1H), 6.86 – 6.77 (m, 2H), 6.53 (s, 1H), 6.41 (dd, 1H), 6.27 (s, 1H), 6.06(dd, 1H), 5.30 (d, 1H), 4.37 (s, 1H), 3.75 (s, 3H), 3.72 (d, 1H), 3.59 (s,3H), 3.50 (d, 2H), 3.36 (s, 1H), 3.25 (s, 4H), 3.15 (s, 1H), 3.10 – 2.98 (m,1H), 2.78 (m, 3H), 2.72 – 2.62 (m, 4H), 2.56 (d, 4H), 2.47 – 2.35 (m, 2H), 2.23 (m, 1H); ESI-MS: m / z 843.20 [M+H] + .

[0052] B-08: Orange solid, yield 84%; 1 H NMR (600 MHz, Chloroform-d) δ 7.75 (d, 2H),7.31 (dd, 1H), 7.17 (d, 1H), 6.85 (dd, 1H), 6.75 (dd, 1H), 6.55 – 6.48 (m,2H), 6.28 (s, 1H), 5.76 (dd, 1H), 5.28 (s, 3H), 4.12 (d, 1H), 3.84 (s, 4H), 3.75 (s, 4H), 3.61 (s, 5H), 3.24 (d, 5H), 3.10 (dd, 1H), 3.00 (dd, 1H), 2.94(s, 1H), 2.87 (s, 1H), 2.72 (dd, 5H), 2.59 (s, 3H), 2.53 (s, 3H), 2.37 (dd,3H), 2.24 (m, 1H); ESI-MS: m / z 793.30 [M+H] + .

[0053] B-09: White solid, yield 73%; 11H NMR (600 MHz, Chloroform-d) δ 7.76 – 7.69(m, 2H), 7.31 (dd, 1H), 7.23 (d, 3H), 7.16 (d, 1H), 6.84 (dd, 1H), 6.76 (dd,1H), 6.53 (s, 1H), 6.42 (dd, 1H), 6.28 (s, 1H), 5.80 (dd, 1H), 5.29 (d, 1H),4.14 (d, 1H), 3.75 (s, 3H), 3.61 (s, 4H), 3.58 (s, 1H), 3.26 (s, 5H), 3.11(dd, 1H), 3.04 – 2.97 (m, 1H), 2.80 – 2.66 (m, 5H), 2.61 (s, 3H), 2.53 (s,3H), 2.45 – 2.33 (m, 7H), 2.26 (m, 1H); ESI-MS: m / z 777.31 [M+H] + .

[0054] B-10-2: White solid, yield 83%; 1 1H NMR (600 MHz, Chloroform-d) δ 7.9 – 7.8(m, 2H), 7.8 – 7.8 (m, 2H), 7.6 (td, 1H), 7.6 – 7.5 (m, 1H), 7.5 – 7.4 (m,4H), 7.3 – 7.3 (m, 1H), 7.2 (d, 1H), 6.8 (dd, 1H), 6.8 (dd, 1H), 6.6 – 6.5(m, 1H), 6.4 (dd, 1H), 6.3 (s, 1H), 5.9 (dd, 1H), 5.3 – 5.3 (m, 1H), 4.1 (d,1H), 3.6 (s, 4H), 3.4 (d, 1H), 3.3 (d, 1H), 3.3 – 3.2 (m, 1H), 3.2 (s, 3H),3.1 (ddt, 2H), 3.1 (dt, 1H), 3.0 (s, 3H), 2.9 – 2.8 (m, 1H), 2.8 – 2.7 (m,1H), 2.7 (tt, 3H), 2.6 (s, 3H), 2.5 (s, 3H), 2.3 (tt, 2H), 2.2 (m, 1H). ESI-MS: m / z 1061.10 [M+H] +.

[0055] B-10: White solid, yield 78%; 1 H NMR (600 MHz, Chloroform-d) δ 7.94 – 7.82(m, 2H), 7.64 – 7.59 (m, 1H), 7.55 – 7.42 (m, 3H), 7.15 (d, 1H), 6.86 – 6.79(m, 2H), 6.53 (s, 1H), 6.40 (dd, 1H), 6.28 (s, 1H), 5.89 (dd, 1H), 5.25 (d,1H), 4.38 (d, 1H), 3.75 (s, 5H), 3.60 (s, 3H), 3.50 (dd, 2H), 3.37 (s, 1H),3.25 (s, 4H), 2.79 (td, 3H), 2.69 (s, 3H), 2.67 – 2.58 (m, 2H), 2.57 (s, 3H), 2.47 – 2.38 (m, 2H), 2.26 (m, 1H); ESI-MS: m / z 843.20 [M+H] + .

[0056] B-11-2: White solid, yield 81%; 11H NMR (600 MHz, Chloroform-d) δ 8.38 (d, 1H), 8.32 (d, 1H), 7.93 – 7.84 (m, 8H), 7.82 (dd, 1H), 7.69 (m, 1H), 7.66 – 7.54 (m, 4H), 7.31 (d, 1H), 7.22 (d, 1H), 6.82 (dd, 1H), 6.64 (dd, 1H), 6.45 (s, 1H), 6.24 (td, 1H), 6.01 (s, 1H), 5.52 – 5.44 (m, 1H), 5.12 (d, 1H), 4.24 (s, 1H), 3.65 (s, 1H), 3.40 (s, 4H), 3.36 (s, 1H), 3.30 – 3.22 (m, 2H), 3.21 (s, 1H), 3.17 (d, 2H), 3.11 (s, 3H), 3.03 (d, 1H), 2.81 (s, 3H), 2.75 (s, 1H), 2.69 (dd, 3H), 2.63 (s, 3H), 2.56 (d, 4H), 2.38 (s, 1H), 2.31 (d, 1H), 2.13 (m, 1H); ESI-MS: m / z 1003.32 [M+H] + .

[0057] B-11: White solid, yield 83%; 11H NMR (600 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.86 (dt, 4H), 7.65 (t, 1H), 7.58 (t, 1H), 7.30 – 7.26 (m, 1H), 7.19 (d, 1H), 6.77 (dd, 1H), 6.59 (dd, 1H), 6.45 (s, 1H), 6.23 (dd, 1H), 6.03 (s, 1H), 5.35 (dd, 1H), 5.27 (s, 1H), 5.14 (d, 1H), 4.21 (d, 1H), 3.89 (s, 1H), 3.71 (s, 3H), 3.65 (d, 1H), 3.45 (s, 3H), 3.36 – 3.30 (m, 2H), 3.20 (s, 3H), 3.16 – 3.08 (m, 2H), 2.69 (md, 4H), 2.60 (s, 3H), 2.53 (s, 3H), 2.39 (td, 1H), 2.32 (dt, 1H), 2.14 (m, 1H), 1.96 (s, 1H); ESI-MS: m / z 813.31 [M+H] + .

[0058] B-12: White solid, yield 76%; 1 1H NMR (600 MHz, Chloroform-d) δ 8.04 (d, 1H), 7.79 – 7.68 (m, 3H), 7.63 (m, 2H), 7.09 (d, 1H), 6.92 (s, 1H), 6.83 (dd, 1H), 6.55 (s, 1H), 6.47 – 6.40 (m, 1H), 6.30 (s, 1H), 6.06 (dd, 1H), 5.33 (d, 1H), 4.46 (d, 1H), 3.80 (d, 1H), 3.76 (s, - 4H), 3.66 – 3.62 (m, 1H), 3.59 (s, 3H), 3.32 (s, 2H), 3.25 (s, 3H), 2.83 (dt, 3H), 2.73 (s, 4H), 2.68 (d, 2H), 2.59 (s, 3H), 2.52 – 2.39 (m, 2H), 2.28 (d, 1H); ESI-MS: m / z 808.28 [M+H] + .

[0059] B-13-2: White solid, yield 69%; 1 H NMR (600 MHz, Chloroform-d) δ 8.36 –8.19 (m, 5H), 8.15 – 7.97 (m, 5H), 7.47 (s, 1H), 7.19 (d, 1H), 6.93 – 6.80(m, 2H), 6.56 (d, 2H), 6.29 (s, 1H), 5.69 (s, 1H), 5.27 (d, 1H), 4.36 (s,1H), 3.77 – 3.68 (m, 1H), 3.61 (s, 3H), 3.49 (d, 1H), 3.41 (d, 1H), 3.30 (s,4H), 3.20 – 3.11 (m, 2H), 3.09 – 3.02 (m, 1H), 2.96 (s, 3H), 2.78 (d, 6H), 2.62 (d, 3H), 2.45 (s, 2H), 2.27 (s, 1H); ESI-MS: m / z 993.26 [M+H] + .

[0060] B-13: White solid, yield 77%; 1 H NMR (600 MHz, Chloroform-d) δ 8.33 – 8.22(m, 2H), 8.09 – 8.01 (m, 2H), 7.51 (d, 1H), 7.19 (d, 1H), 6.90 – 6.77 (m,2H), 6.60 – 6.48 (m, 2H), 6.29 (s, 1H), 5.61 (dd, 1H), 5.29 (d, 1H), 4.32 (s,1H), 3.76 (d, 3H), 3.69 (s, 1H), 3.59 (s, 3H), 3.48 (d, 2H), 3.37 (s, 1H),3.25 (s, 3H), 3.20 (d, 2H), 2.80 – 2.72 (m, 4H), 2.68 (s, 3H), 2.57 (s, 4H), 2.43 (t, 2H), 2.28 – 2.20 (m, 1H); ESI-MS: m / z 808.28 [M+H] + .

[0061] B-14-2: White solid, yield 84%; 1H NMR (600 MHz, Chloroform-d) δ 7.68 (s,2H), 7.63 (s, 2H), 7.57 (s, 5H), 7.30 (dd, 1H), 7.20 (d, 1H), 6.85 (dd, 1H),6.77 (dd, 1H), 6.53 (s, 1H), 6.49 (dd, 1H), 6.29 (s, 1H), 5.67 (s, 1H), 5.25(s, 1H), 4.15 (d, 1H), 3.63 (s, 3H), 3.45 (s, 1H), 3.39 (d, 1H), 3.30 (s,4H), 3.17 – 3.07 (m, 3H), 2.99 (s, 3H), 2.95 – 2.89 (m, 1H), 2.76 – 2.68 (m,3H), 2.62 (s, 3H), 2.54 (s, 3H), 2.39 – 2.34 (m, 2H), 2.21 (td, 1H); ESI-MS: m / z 1061.10 [M+H] + .

[0062] B-14: White solid, yield 86%; 1 H NMR (600 MHz, Chloroform-d) δ 7.75 (d, 1H),7.67 (d, 2H), 7.57 (t, 3H), 7.16 (s, 1H), 6.84 (d, 2H), 6.55 – 6.49 (m, 2H),6.30 (s, 1H), 5.69 (s, 1H), 5.26 (d, 1H), 4.43 (d, 1H), 3.74 (d, 4H), 3.62(s, 3H), 3.52 (d, 2H), 3.36 (s, 2H), 3.24 (s, 4H), 3.16 – 3.11 (m, 1H), 2.89– 2.76 (m, 4H), 2.74 (s, 4H), 2.59 (s, 4H), 2.52 – 2.40 (m, 2H), 2.26 – 2.19(m, 1H); ESI-MS: m / z 843.20 [M+H] + .

[0063] B-15: White solid, yield 83%; 1H NMR (600 MHz, Chloroform-d) δ 7.64 – 7.53(m, 1H), 7.37 (m, 1H), 7.17 – 7.09 (m, 2H), 6.96 – 6.83 (m, 4H), 6.58 (d,1H), 6.34 – 6.26 (m, 2H), 5.50 (d, 1H), 4.39 (d, 1H), 3.76 (s, 3H), 3.73 (s,1H), 3.61 (s, 3H), 3.50 (d, 1H), 3.36 (d, 1H), 3.33 – 3.29 (m, 2H), 3.27 (s,3H), 3.23 (dt, 2H), 3.19 (s, 1H), 2.85 (dd, 2H), 2.74 (dt, 2H), 2.70 (s, 3H), 2.64 (d, 1H), 2.57 (s, 3H), 2.47 – 2.38 (m, 2H), 2.33 (m, 1H), 1.96 (dp, 3H),1.25 – 1.18 (m, 23H); ESI-MS: m / z 855.45 [M+H] + .

[0064] Example 3 A method for preparing a berberine derivative includes the following steps: Steps (1)-(4) of Example 3 are the same as steps (1)-(4) of Example 1; (5) Take 100 mg of dried intermediate 4 and 68 µL of triethylamine in a 10 mL round-bottom flask, add 2 mL of DCM under Ar protection and stir to dissolve the sample. Add isocyanate reagent at 0 °C and place at room temperature after 30 min. The reaction was detected by TLC and was completed in 1-2 h. Pour the reaction solution into ice water, adjust the pH to alkaline with saturated NaHCO3 aqueous solution, extract with DCM 3 times, wash with saturated NaCl solution, dry with anhydrous sodium sulfate, filter and combine the organic phases, and recover the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography with DCM:MeOH = 50:1, v / v to obtain the berberine derivative.

[0065] In step (5), different isocyanate reagents are added, resulting in different products.

[0066] Take general formula 3 as an example: The specific isocyanate reagents and their corresponding compound structures are shown in Table 3: Table 3. R5 and R6 groups corresponding to different reagents in Formula 3

[0067] Spectral data of each substance: C-01: White solid, yield 86%; 1 H NMR (600 MHz, Chloroform-d) δ 7.86 – 7.72(m, 2H), 7.48 (dd, 1H), 7.16 (dd, 1H), 6.88 (d, 2H), 6.85 – 6.79 (m, 2H),6.55 – 6.47 (m, 2H), 6.28 (s, 1H), 5.83 (dd, 1H), 5.29 – 5.25 (m, 1H), 4.33 (d, 1H), 3.84 (s, 4H), 3.75 (s, 3H), 3.71 (t, 1H), 3.61 (s, 3H), 3.49 – 3.42(m, 2H), 3.25 (s, 3H), 3.22 – 3.17 (m, 1H), 2.82 – 2.69 (m, 5H), 2.67 (s,3H), 2.57 (d, 4H), 2.43 (t, 2H), 2.28 – 2.22 (m, 1H), 1.98 (d, 1H); ESI-MS: m / z 793.30 [M+H] + .

[0068] C-02: White solid, yield 76%; 11H NMR (600 MHz, Chloroform-d) δ 7.91 (d, 2H), 7.83 (d, 1H), 7.65 (d, 1H), 7.47 – 7.43 (m, 2H), 7.42 – 7.33 (m, 2H), 7.02 – 6.91 (m, 2H), 6.91 – 6.83 (m, 1H), 6.75 – 6.67 (m, 2H), 6.67 – 6.62 (m, 1H), 6.36 – 6.26 (m, 2H), 5.39 (d, 1H), 4.30 (dd, 1H), 3.76 (d, 3H), 3.64 – 3.55 (m, 3H), 3.41 (s, 1H), 3.29 (d, 2H), 3.19 (d, 4H), 3.14 – 3.05 (m, 2H), 3.04 – 2.87 (m, 2H), 2.85 – 2.71 (m, 4H), 2.65 (d, 1H), 2.61 – 2.51 (m, 5H), 2.44 – 2.32 (m, 3H); ESI-MS: m / z 792.35 [M+H] + .

[0069] C-03: White solid, yield 81%; 1 1H NMR (600 MHz, Chloroform-d) δ 7.86 (d, 2H), 7.63 (s, 2H), 7.59 (s, 2H), 7.49 (d, 2H), 7.42 (s, 2H), 7.28 (s, 1H), 6.84 (s, 1H), 6.70 (d, 1H), 6.50 (s, 2H), 6.11 (s, 1H), 5.55 (s, 1H), 5.22 (d, 1H), 4.26 (s, 1H), 3.73 (s, 3H), 3.67 (s, 1H), 3.40 (s, 3H), 3.26 (s, 3H), 3.22 (td, 3H), 3.17 (dd, 3H), 2.73 (m, 4H), 2.64 (s, 3H), 2.6 – 2.51 (m, 5H), 2.41 (s, 2H), 2.26 (s, 1H); ESI-MS: m / z 818.37 [M+H] + .

[0070] C-04: White solid, yield 62%; 1 H NMR (600 MHz, Chloroform-d) δ 7.32 (s,1H), 6.93 (dd, 2H), 6.88 (dd, 1H), 6.85 – 6.76 (m, 2H), 6.60 (d, 1H), 6.32(s, 2H), 6.10 (s, 1H), 5.49 (s, 1H), 4.78 (s, 1H), 4.27 (d, 1H), 3.73 (s,4H), 3.62 (d, 4H), 3.39 (d, 2H), 2.92 – 2.81 (m, 4H), 2.77 – 2.69 (m, 4H),2.63 (s, 4H), 2.59 – 2.49 (m, 5H), 2.46 – 2.35 (m, 3H), 1.57 – 1.45 (m, 5H);ESI-MS: m / z 708.35 [M+H] + .

[0071] C-05-02: White solid, yield 69%; 1 H NMR (600 MHz, Chloroform-d) δ 7.37 (dd,1H), 7.03 (d, 1H), 6.98 – 6.93 (m, 2H), 6.86 (m, 1H), 6.79 (dd, 1H), 6.64 (s,1H), 6.34 (d, 2H), 5.69 (d, 1H), 5.50 (d, 1H), 5.13 (d, 1H), 4.49 (d, 1H), 4.34 – 4.20 (m, 1H), 3.74 (s, 4H), 3.61 (s, 4H), 3.43 – 3.34 (m, 2H), 3.29(d, 1H), 3.23 (s, 3H), 3.19 – 3.16 (m, 2H), 3.15 – 3.08 (m, 2H), 3.03 – 2.96(m, 1H), 2.95 – 2.84 (m, 4H), 2.73 (m, 4H), 2.64 (s, 4H), 2.55 (d, 4H), 2.50(s, 1H), 2.40 (td, 4H), 2.17 (s, 3H); ESI-MS: m / z 821.44 [M+H] + .

[0072] C-05: White solid, yield 72%;1 H NMR (600 MHz, Methanol-d4) δ 7.78 – 7.70(m, 1H), 7.30 – 7.22 (m, 2H), 7.17 (m, 1H), 7.06 – 6.99 (m, 2H), 6.97 – 6.92(m, 1H), 6.83 (d, 1H), 6.68 (dd, 1H), 5.66 (d, 1H), 4.78 (dd, 1H), 4.68 (d,1H), 4.32 (s, 1H), 4.16 – 4.07 (m, 1H), 4.01 (d, 3H), 3.93 (s, 2H), 3.83 (d,1H), 3.67 (s, 1H), 3.62 (dd, 1H), 3.53 (d, 1H), 3.51 (s, 2H), 3.48 – 3.42 (m,1H), 3.41 (s, 1H), 3.37 – 3.33 (m, 1H), 3.24 (dd, 1H), 3.20 – 3.16 (m, 1H),3.11 – 3.04 (m, 2H), 2.97 (d, 3H), 2.94 – 2.88 (m, 4H), 1.64 (d, 9H); ESI-MS:m / z 722.37 [M+H] + .

[0073] C-06: White solid, yield 67%; 1H NMR (600 MHz, Chloroform-d) δ 10.77 –10.50 (m, 1H), 8.62 (s, 1H), 7.51 – 7.19 (m, 1H), 7.00 – 6.84 (m, 2H), 6.79 –6.71 (m, 1H), 6.64 (p, 1H), 6.48 – 6.28 (m, 2H), 5.43 (s, 1H), 5.28 (s, 1H), 4.38 – 4.10 (m, 3H), 3.78 (d, 3H), 3.66 – 3.53 (m, 2H), 3.54 – 3.43 (m, 1H),3.41 – 3.28 (m, 2H), 3.25 (d, 3H), 3.17 – 3.06 (m, 2H), 3.06 – 2.87 (m, 2H), 2.87 – 2.72 (m, 3H), 2.71 – 2.51 (m, 5H), 2.49 – 2.32 (m, 3H), 1.38 – 0.95(m, 5H).; ESI-MS: m / z 822.25 [M+H] + .

[0074] Example 4 A method for preparing a berberine derivative includes the following steps: Steps (1)-(4) of Example 4 are the same as steps (1)-(4) of Example 1; (5) Take 100 mg of dried intermediate 4 and 68 µL of triethylamine in a 10 mL round-bottom flask, add 2 mL of DCM under Ar protection and stir to dissolve the sample. Add isothiocyanate reagent at 0 °C and place at room temperature after 30 min. The reaction was detected by TLC and was completed in 1-2 h. Pour the reaction solution into ice water, adjust the pH to alkaline with saturated NaHCO3 aqueous solution, extract with DCM 3 times, wash with saturated NaCl solution, dry with anhydrous sodium sulfate, filter and combine the organic phases, and recover the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography with DCM:MeOH = 50:1, v / v to obtain the berberine derivative.

[0075] In step (5), different isothiocyanate reagents are added, resulting in different products.

[0076] Take general formula 4 as an example: , The specific isothiocyanate reagents and their corresponding compound structures are shown in Table 4: Table 4. R7 groups corresponding to different reagents in Formula 4

[0077] The spectral data for each substance are as follows: D-01: White solid, yield 82%; 1 H NMR (600 MHz, Chloroform-d) δ 10.77 –10.50 (m, 1H), 8.62 (s, 1H), 7.51 – 7.19 (m, 1H), 7.00 – 6.84 (m, 2H), 6.79 –6.71 (m, 1H), 6.64 (p, 1H), 6.48 – 6.28 (m, 2H), 5.43 (s, 1H), 5.28 (s, 1H), 4.38 – 4.10 (m, 3H), 3.78 (d, 3H), 3.66 – 3.53 (m, 2H), 3.54 – 3.43 (m, 1H),3.41 – 3.28 (m, 2H), 3.25 (d, 3H), 3.17 – 3.06 (m, 2H), 3.06 – 2.87 (m, 2H), 2.87 – 2.72 (m, 3H), 2.71 – 2.51 (m, 5H), 2.49 – 2.32 (m, 3H), 1.38 – 0.95(m, 5H); ESI-MS: m / z 754.3 [M+H] + .

[0078] D-02: White solid, yield 79%; 11H NMR (600 MHz, Chloroform-d) δ 7.35 (dd, 1H), 6.98 – 6.91 (m, 3H), 6.77 – 6.72 (m, 2H), 6.63 (d, 1H), 6.33 (d, 2H), 5.67 (s, 1H), 5.38 (d, 1H), 4.26 (d, 2H), 3.74 (d, 4H), 3.62 (s, 4H), 3.37 (s, 2H), 3.31 (d, 1H), 3.20 (s, 4H), 3.14 (d, 2H), 3.11 – 3.00 (m, 2H), 2.96 – 2.84 (m, 3H), 2.84 – 2.73 (m, 3H), 2.71 – 2.60 (m, 7H), 2.56 (d, 4H), 2.47 – 2.34 (m, 4H), 2.01 (s, 2H), 1.63 – 1.56 (m, 3H), 1.43 – 1.37 (m, 2H), 1.11 (d, 3H); ESI-MS: m / z 764.36 [M+H] + .

[0079] D-03: White solid, yield 68%; 1 1H NMR (600 MHz, Chloroform-d) δ 7.33 (dd, 1H), 6.96 – 6.90 (m, 3H), 6.75 – 6.70 (m, 2H), 6.62 (s, 1H), 6.33 (d, 2H), 5.82 (s, 1H), 5.42 – 5.35 (m, 1H), 4.27 (d, 1H), 3.76 (d, 4H), 3.69 – 3.64 (m, 1H), 3.63 (s, 4H), 3.55 (dt, 1H), 3.36 (d, 1H), 3.31 (d, 1H), 3.18 (d, 4H), 3.12 (dd, 1H), 3.07 – 2.99 (m, 1H), 2.92 – 2.86 (m, 2H), 2.81 – 2.72 (m, 3H), 2.71 – 2.64 (m,3H), 2.61 (d, 3H), 2.55 (d, 4H), 2.48 – 2.34 (m, 4H), 1.51 (d, 2H), 1.35 – 1.23 (m, 3H); ESI-MS: m / z 738.35 [M+H]+ .

[0080] D-04: White solid, yield 77%; 1 H NMR (600 MHz, Methanol-d4) δ 8.20 (dd, 2H),8.09 – 7.95 (m, 2H), 7.33 (dd, 1H), 7.00 (t, 2H), 6.93 – 6.89 (m, 1H), 6.78(dq, 1H), 6.76 – 6.72 (m, 1H), 6.70 – 6.64 (m, 1H), 6.56 (d, 1H), 6.48 – 6.39(m, 1H), 5.40 (d, 1H), 4.56 – 4.41 (m, 1H), 4.16 – 4.03 (m, 1H), 3.82 (d,3H), 3.67 (s, 2H), 3.64 – 3.55 (m, 2H), 3.42 (s, 2H), 3.39 – 3.33 (m, 1H), 3.28 (s, 3H), 3.24 – 3.16 (m, 3H), 3.12 – 3.02 (m, 2H), 2.87 (m, 4H), 2.73(d, 3H), 2.68 – 2.59 (m, 6H), 2.50 – 2.43 (m, 1H); ESI-MS: m / z 803.30 [M+H] + .

[0081] D-05: White solid, yield 74%; 1H NMR (600 MHz, Chloroform-d) δ 7.32 (dd,2H), 6.99 (s, 1H), 6.88 (s, 4H), 6.79 (s, 1H), 6.73 (s, 1H), 6.59 (m, 2H),6.37 (dd, 1H), 6.31 (s, 1H), 5.28 (s, 1H), 4.41 (s, 1H), 3.95 (d, 1H), 3.79(m, 3H), 3.77 (d, 3H), 3.73 (d, 1H), 3.59 (dd, 2H), 3.49 (s, 1H), 3.34 (m,2H), 3.27 (s, 1H), 3.21 (d, 2H), 3.11 (s, 3H), 2.97 (d, 1H), 2.85 (s, 1H), 2.78 (s, 3H), 2.62 (s, 2H), 2.56 (m, 6H), 2.35 (s, 1H); ESI-MS: m / z 788.32 [M+H] + .

[0082] D-06: Beige solid, yield 82%; 1 H NMR (600 MHz, Chloroform-d) δ 7.94 – 7.82(m, 2H), 7.64 – 7.59 (m, 1H), 7.55 – 7.42 (m, 3H), 7.15 (d, 1H), 6.86 – 6.79(m, 2H), 6.53 (s, 1H), 6.40 (dd, 1H), 6.28 (s, 1H), 5.89 (dd, 1H), 5.25 (d,1H), 4.38 (d, 1H), 3.75 (m, 5H), 3.60 (s, 3H), 3.50 (dd, 2H), 3.37 (s, 1H),3.25 (s, 4H), 2.79 (td, 3H), 2.69 (s, 3H), 2.67 – 2.58 (m, 2H), 2.57 (s, 3H), 2.47 – 2.38 (m, 2H), 2.26 (m, 1H); ESI-MS: m / z 695.32 [M+H] + .

[0083] D-07: Yellow solid, yield 82%; 1H NMR (600 MHz, Methanol-d4) δ 7.66 – 7.44(m, 3H), 7.36 (t, 2H), 7.07 – 6.98 (m, 2H), 6.97 – 6.90 (m, 1H), 6.85 – 6.81(m, 1H), 6.77 (d, 1H), 6.70 (d, 1H), 6.61 (d, 1H), 6.44 (dd, 1H), 4.68 – 4.48(m, 1H), 4.31 (s, 1H), 3.83 (d, 3H), 3.76 (q, 1H), 3.67 (s, 1H), 3.42 (s,2H), 3.38 – 3.32 (m, 1H), 3.29 (s, 5H), 3.23 – 3.09 (m, 3H), 2.99 (q, 2H), 2.95 – 2.87 (m, 3H), 2.85 (d, 2H), 2.81 – 2.75 (m, 1H), 2.72 (t, 4H), 2.66(s, 1H), 2.52 (d, 1H);. ESI-MS: m / z 792.27 [M+H] + .

[0084] D-08: White solid, yield 83%. 1H NMR (600 MHz, Methanol- d4) δ 7.78 – 7.70 (m,1H), 7.30 – 7.22 (m, 2H), 7.17 (m, 1H), 7.06 – 6.99 (m, 2H), 6.97 – 6.92 (m,1H), 6.83 (d, 1H), 6.68 (dd, 1H), 5.66 (d, 1H), 4.78 (dd, 1H), 4.68 (d, 1H), 4.32 (s, 1H), 4.16 – 4.07 (m, 1H), 4.01 (d, 3H), 3.93 (s, 2H), 3.83 (d, 1H), 3.67 (s, 1H), 3.62 (dd, 1H), 3.53 (d, 1H), 3.51 (s, 2H), 3.48 – 3.42 (m, 1H), 3.41 (s, 1H), 3.37 – 3.33 (m, 1H), 3.24 (dd, 1H), 3.20 – 3.16 (m, 1H), 3.11 –3.04 (m, 2H), 2.97 (d, 3H), 2.94 – 2.88 (m, 4H), 1.64 (d, 9H); ESI-MS: m / z696.30 [M+H] + .

[0085] D-09: White solid, yield 84%; 1 H NMR (600 MHz, Chloroform-d) δ 7.96 (s,2H), 7.64 (s, 1H), 7.54 (s, 2H), 7.39 (s, 1H), 7.02 (s, 1H), 6.91 (s, 2H),6.80 (s, 1H), 6.64 (s, 2H), 6.41 (s, 1H), 6.34 (s, 1H), 5.36 (s, 1H), 4.40(s, 1H), 3.80 (s, 3H), 3.63 (s, 2H), 3.40 (s, 3H), 3.27 (s, 3H), 3.09 (s,3H), 2.79 (s, 5H), 2.62 (s, 6H), 2.54 (s, 3H), 2.39 (s, 1H); ESI-MS: m / z786.31 [M+H] + .

[0086] D-10: Yellow solid, yield 87%; 1H NMR (600 MHz, Methanol-d4) δ 7.6 – 7.4 (m,9H), 7.4 – 7.3 (m, 1H), 7.0 (t, 4H), 6.9 (tt, 2H), 6.8 – 6.8 (m, 2H), 6.8 (d,1H), 6.7 (d, 2H), 6.6 (d, 2H), 6.5 – 6.4 (m, 2H), 5.4 (d, 1H), 4.6 – 4.4 (m,2H), 4.2 (s, 1H), 3.9 – 3.9 (m, 1H), 3.8 (d, 6H), 3.7 – 3.6 (m, 6H), 3.4 (s,5H), 3.3 (s,5H), 3.3 – 3.1 (m, 6H), 3.1 – 3.0 (m, 3H), 2.9 – 2.8 (m, 7H), 2.8 – 2.7 (m, 7H), 2.7 (q, 12H), 2.5 (d, 1H), 1.4 – 1.2 (m, 3H); ESI-MS: m / z 838.22 [M+H] + .

[0087] D-11: Yellow solid, yield 70%; 1 H NMR (600 MHz, Chloroform-d) δ 7.37 (s, 1H),7.28 (t, 1H), 7.02 – 6.96 (m, 1H), 6.91 – 6.83 (m, 3H), 6.76 (d, 1H), 6.62(d, 2H), 6.44 – 6.28 (m, 3H), 5.34 – 5.22 (m, 1H), 4.38 (d, 2H), 3.95 – 3.87(m, 1H), 3.79 (d, 4H), 3.61 (s, 3H), 3.55 (s, 1H), 3.38 – 3.34 (m, 2H), 3.22(s, 3H), 3.13 (d, 3H), 2.99 – 2.89 (m, 4H), 2.64 – 2.59 (m, 7H), 2.42 – 2.35(m, 1H); ESI-MS: m / z 826.24 [M+H] + .

[0088] Example 5 Application of berberine derivatives in the preparation of anti-breast cancer drugs The inhibitory activity of berberine derivatives against four breast cancer cell lines (MDA-MB-231, MDA-MB-468, 4T1, and MCF-7) and one normal breast epithelial cell line (MCF-10A) was determined using the MTT assay. The specific method is as follows: ① Take MDA-MB-231, MDA-MB-468, 4T1, MCF-7, and MCF-10A cells in logarithmic growth phase, prepare cell suspensions using standard passage methods, and adjust the concentration to 0.8 × 10⁻⁶ cells after cell counting. 5 Cells / well; after thorough mixing, use a pipette to add 190 μL of cell suspension to a 96-well plate, add 100 μL of PBS to the edge wells to prevent evaporation and not to participate in the experiment; incubate at 37℃, 5% CO2 for 24 h, and then set aside.

[0089] ② After 24 h, add 10 µL of culture medium containing gradient concentrations of compounds from Examples 1-4 to each well, with final concentrations of 1.25, 2.5, 5, 7.5, and 10 µM. Each concentration is tested in triplicate. A cell control group is also included. After mixing, continue culturing for 48 h. Afterward, add 20 µL of 5 mg / mL MTT solution and incubate at 37°C for 4 h. Discard the supernatant, centrifuge at 1000 rpm for 5–10 min, add 150 µL of DMSO to dissolve the crystals, and gently shake for 10 min to mix. Measure the absorbance at 490 nm using a microplate reader (A) and calculate the cell inhibition rate and selectivity index (SI). Cell inhibition rate = (1 - A experimental group) / A control group × 100%.

[0090] The results are shown in Table 5. Table 5. In vitro anti-breast cancer inhibitory activity of berberine derivatives.

[0091] Among the 56 berberine derivatives synthesized using intermediate 4, the in vitro inhibitory activity against breast cancer showed that compound B-12 exhibited the best activity against MDA-MB-231 and 4T1 cells, with an IC50 concentration of [missing value]. 50 The values ​​reached 2.23±0.1 and 1.51±0.33 µM, respectively.

[0092] Example 6 Flow cytometry detection of apoptosis Compound B-12 was used in the experiments, and the experimental cell lines were MDA-MB-231 and 4T1 cells.

[0093] The experimental groups were set up as follows: blank control group, B-12 experimental group (1.25, 2.5, 5 µM) and positive control group (berberine, 5 µM).

[0094] The specific steps are as follows: Dilute 10× Binding buffer with sterile deionized water at a ratio of 1:9 to prepare 1× working solution. Take MDA-MB-231 and 4T1 cells in the logarithmic growth phase, moderately digest them with EDTA-free trypsin, add culture medium, and collect the cells by pipetting. Centrifuge at 1000 rpm for 3 min. Wash the cells twice with pre-cooled PBS and resuspend them in 1× Binding buffer to a final volume of 1×10⁻⁶. 6 cell / mL. Take 100µL of cell suspension (1×10⁻⁶ cells / mL). 5 Add 100 cells to an EP tube, add 5 µL Annexin V-FITC and 5 µL PI, incubate at room temperature in the dark for 15 min, then add 400 µL 1× Binding buffer and mix well. Perform flow cytometry analysis within 1 hour. Results are as follows: Figure 1 As shown.

[0095] from Figure 1 It can be seen that after compound B-12 was applied to tumor cells for 24 h, the detection results showed that the early apoptosis rate of MDA-MB-231 cells was significantly increased, and the apoptosis-inducing effect of 2.5 µM compound B-12 on this cell was better than that of berberine. In 4T1 cells, the late apoptosis rate of cells increased in a concentration-dependent manner with the increase of compound B-12 concentration, and its pro-apoptotic effect was also better than that of berberine.

[0096] Example 7 animal experiments A mouse tumor model was established, and 4T1-Luc cells (1×10⁻⁶) were selected. 7 (Number of cells) were transplanted into the axillae of female nude mice weighing approximately 18-20 g, 7 days after transplantation. After successful modeling, on days 0, 3, 6, 9, 12, 15, and 18, mice were administered compound B-12 (10 mg / kg, 100 µL), berberine (10 mg / kg, 100 µL), cisplatin (5 mg / kg, 100 µL), and a control group (0.9% NaCl, 100 µL) via tail vein injection. Mice were sacrificed on day 21, and tumors were dissected and photographed. The results are shown below. Figure 2 As shown.

[0097] Mice were euthanized, tumors were removed, and mice were classified into groups. Figure 2 The results showed that the tumor volume in the drug group (B-12 10 mg / kg) was significantly smaller than that in the control group, and its tumor-suppressing effect was superior to that in the berberine group (Ber 10 mg / kg).

[0098] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A berberine derivative, characterized in that: Includes the following general structural formulas: ,or ,or ,or .

2. The berberine derivative according to claim 1, characterized in that: In general formula 1, R1 is selected from: , , , , , , , , , , and , The R2 is selected from hydrogen atoms, , , and .

3. The berberine derivative according to claim 1, characterized in that: In general formula 2, R3 is selected from: , , , , , , , , , , , , , and The R4 is selected from hydrogen atoms, , , , and .

4. The berberine derivative according to claim 1, characterized in that: In general formula 3, R5 is selected from: , , , , , and The R6 is selected from hydrogen atoms and .

5. The berberine derivative according to claim 1, characterized in that: In general formula 4, R7 is selected from: , , , , , , , , , and .

6. A method for preparing the berberine derivative according to claim 1, characterized in that: Includes the following steps: (1) Weigh berberine and place it in a reaction vessel. Dissolve it in dry dichloromethane and add triethylamine to obtain reaction system 1. Slowly add acetyl chloride at 0–5℃ for 30–40 min. Wash with saturated sodium bicarbonate and extract with dichloromethane 2–3 times. Combine the organic phases, dry and filter, and concentrate under reduced pressure to obtain intermediate 1. Under argon protection, dissolve intermediate 1 in dry dichloromethane to obtain reaction system 2. Add nitric anhydride at 0–5℃ and react. Then wash with saturated sodium bicarbonate and extract with dichloromethane 2–3 times. Combine the organic phases, dry and filter, and concentrate under reduced pressure to obtain intermediate 2. (2) Take intermediate 2 and sodium methoxide and dissolve them in methanol. Reflux the reaction mixture in an oil bath at 40–50 °C. After the reaction mixture is concentrated under reduced pressure, dissolve it in dichloromethane. Wash the mixture with saturated sodium chloride aqueous solution and extract it with dichloromethane 2–3 times. Dry the organic phase, filter it, and concentrate it under reduced pressure to obtain intermediate 3. (3) Intermediate 3 and 5% palladium on carbon were placed in methanol, and hydrazine hydrate was added dropwise and refluxed in an oil bath at 70-80℃. The reaction solution was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, dissolved in dichloromethane, washed with saturated sodium chloride aqueous solution and extracted 2-3 times, the organic phase was dried, filtered and concentrated under reduced pressure to obtain intermediate 4. (4) Intermediate 4 and triethylamine were dissolved in dichloromethane under argon protection. The reaction reagent was added at 0°C and reacted for 30 min. The reaction was then carried out at room temperature for 1–2 h. The reaction solution was poured into ice water, adjusted to alkalinity with saturated NaHCO3, extracted with dichloromethane 2–3 times, washed with saturated NaCl, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain a solid. After purification, berberine derivatives were obtained. The reaction reagents are selected from acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates; The mass ratio of intermediate 4 to the reaction reagent is 1:1-1.

2.

7. The method for preparing the berberine derivative according to claim 6, characterized in that: The amount of intermediate 1 and nitroacetic anhydride used in step (1) is: 3 mL of nitroacetic anhydride for every 1 g of intermediate 1.

8. The method for preparing the berberine derivative according to claim 6, characterized in that: The mass ratio of intermediate 2 to sodium methoxide in step (2) is 3-5:

1.

9. The method for preparing the berberine derivative according to claim 6, characterized in that: The mass ratio of intermediate 3 to palladium on carbon in step (3) is 5-7:

1.

10. The use of a berberine derivative or its salt, or a formulation containing a berberine derivative, in the preparation of an anti-breast cancer drug, characterized in that: The berberine derivative is the berberine derivative according to any one of claims 1-5.

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