Preparation and application of novel (3, 10-dimethoxy-12, 13-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride)-dehydroberberine derivative

Abstract

The invention relates to a novel (3, 10-dimethoxy-12, 13-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride)-dehydroberberine derivative DH-Ber-12 and an application thereof, and designs a brand new artificially modified dehydroberberine derivative, in particular to a novel (3, 10-dimethoxy-12, 13-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride)-dehydroberberine derivative. In the berberine derivative, 3, 9-dimethoxy-5, 6-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride (Ber-12) shows good performance in the aspects of transcriptional activation activity, solubility and colon cancer cell inhibition activity of RXR [alpha], and the performance of the berberine derivative is expected to be improved through structural modification of the berberine derivative. The invention discloses a novel (3, 10-dimethoxy-12, 13-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride)-dehydroberberine derivative DH-Ber-12 and a preparation method of the novel (3, 10-dimethoxy-12, 13-dihydroisoquinolo [3, 2-a] isoquinoline-7-hydrochloride)-dehydroberberine derivative, and belongs to the technical field of medicinal chemistry. The key points of the technical scheme are as follows: the synthesis route is short, the operation is simple and easy, the product structure is novel, the performance is stronger than that of Ber-12, and the method is worthy of further popularization and research.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and mainly relates to the preparation and application of novel (3,10-dimethoxy-12,13-dihydroisoquinolino[3,2-a]isoquinolino-7-chloride)-dehydroberberine derivatives.

[0002] This invention relates to a novel artificially modified berberine derivative, DH-Ber-12, which exhibits superior antitumor activity, higher cancer cell killing activity, better water solubility, and stronger fluorescence. Technical Background

[0003] Berberine (Ber), also known as berberine alkaloid, is a quaternary ammonium isoquinoline alkaloid isolated from the traditional Chinese medicines Coptis chinensis, Phellodendron chinense, and Berberis vulgaris. Its free basic form (berberine hydroxide) is unstable, but exhibits strong stability when bound to hydrochloric acid. In plants, it mainly exists in the form of its salt. Berberine has multi-target pharmacological effects, playing an important role not only in antibacterial and anti-inflammatory activity but also in antitumor activity and metabolic regulation. It has a broad antibacterial spectrum, showing significant inhibitory effects against both Gram-negative and Gram-positive bacteria. It can also exert antitumor effects by regulating apoptosis and inhibiting tumor angiogenesis. In addition, berberine also possesses antibacterial, anticancer, anti-inflammatory, lipid-lowering, blood sugar-lowering, neuroprotective, and cholesterol-lowering effects, and has been used in traditional Chinese medicine and Ayurveda for 3000 years. In the past decade, berberine has received increasing attention as an antitumor drug.

[0004] Berberine has sparked an international research boom due to its natural source, wide range of pharmacological functions, and high safety. However, it also has certain drawbacks: 1) Berberine has very low water and lipid solubility, resulting in low bioavailability and poor efficacy. This is because berberine's near-planar structure and the stacking of aromatic rings lead to poor solubility in water and organic solvents; 2) While berberine itself is not harmful to the human body, the chloride ions in berberine hydrochloride, currently used clinically, pose a potential hazard (the hydrochloric acid produced by the hydrolysis of berberine hydrochloride in the gastrointestinal tract can irritate the gastrointestinal tract and cause discomfort); 3) Berberine is often used to treat gastrointestinal diseases (such as diarrhea and gastroenteritis), with small doses, short treatment durations, and insidious adverse reactions. Long-term use requires consideration of the potential risks associated with excessive accumulation of chloride ions in berberine hydrochloride.

[0005] Research on modifying the structure of berberine dates back to the 1970s. Currently, the focus is mainly on enhancing the affinity of berberine for DNA, thereby improving its anti-tumor activity. For example, literature reports that modifying the C9-0 position with different types of groups can significantly improve the binding ability of berberine to DNA. However, most studies have not monitored the antitumor activity of these derivatives. Therefore, modifying the structure of berberine holds promise for increasing its antitumor activity. Furthermore, introducing a lipophilic group at the C9-0 position enhances its binding ability to the cell membrane, resulting in a 103-fold increase in its ability to inhibit tumor cell growth in vitro. However, this also reduces the water solubility and bioavailability of berberine, which is detrimental to its antitumor effect in vivo.

[0006] In summary, berberine is inexpensive and readily available in clinical practice, and has a history of application in my country spanning thousands of years. Modern pharmacological studies have revealed its diverse pharmacological activities. Recent research indicates that berberine holds great promise in medical imaging, tumor treatment, and antibacterial and anti-inflammatory applications. Therefore, designing higher-quality, more potent berberine for clinical use is of significant practical importance. Summary of the Invention

[0007] The novel (3,10-dimethoxy-12,13-dihydroisoquinolino[3,2-a]isoquinolino-7-hydrochloride)-dehydroberberine derivative is:

[0008]

[0009] 3,10-Dimethoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 7), with the following structural formula:

[0010]

[0011] 3-Fluoro,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 8), with the following structural formula:

[0012]

[0013] 3-Difluoromethyl,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 9), with the following structural formula:

[0014]

[0015] 3-Trifluoromethyl,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 10), with the following structural formula:

[0016]

[0017] 3-Difluoromethoxy,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 11), with the following structural formula:

[0018]

[0019] 3-Trifluoromethoxy,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 12), with the following structural formula:

[0020]

[0021] 3-Methoxy,10-fluoro-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 13), with the following structural formula:

[0022]

[0023] 3-Methoxy,10-difluoromethyl-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 14), with the following structural formula:

[0024]

[0025] 3-Methoxy,10-trifluoromethyl-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 15), with the following structural formula:

[0026]

[0027] 3-Methoxy,10-difluoromethoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 16), with the following structural formula:

[0028]

[0029] 3-Difluoromethoxy,10-methoxy-12,13-dihydroisoquinolino[3,2-a]isoquinoline hydrochloride (compound 17), with the following structural formula:

[0030]

[0031] The dehydroberberine derivative DH-Ber-12 is used in the preparation of antitumor drugs, including those for colorectal cancer.

[0032] The present invention modifies berberine to obtain DH-Ber-12, which is superior to berberine in terms of fluorescence performance, solubility, and inhibitory activity against colorectal cancer cells.

[0033] Experimental results showed that the fluorescence emission and absorption wavelengths of DH-Ber-12 were 1.5 to 2 times greater than those of berberine. DH-Ber-12 was 9 to 10 times more lipid-soluble and 5 to 7 times more water-soluble than berberine. HCT116 cell viability assays confirmed that DH-Ber-12 had a cancer cell lethality 10 to 11 times that of berberine, with a median lethal concentration (LD50) of 12 to 15 μmol / mL, which is 1 / 10 to 1 / 5 of that of berberine.

[0034] Like berberine, DH-Ber-12 inhibits the proliferation of colon cancer cells but has no inhibitory effect on NCM460 colon epithelial cells. This indicates that, after modification, DH-Ber-12 still retains the tumor selectivity of berberine.

[0035] Therefore, it can be seen that the bioavailability of DH-Ber-12 is far superior to that of berberine.

[0036] Literature reports that berberine can act as a novel agonist targeting RXRα to inhibit the proliferation of colon cancer cells; and selectively inhibit the growth of colon cancer cells through RXRα mediation, with minimal impact on normal intestinal epithelial cells. Compared with the natural ligand 9-cis-RA of RXRα used in clinical cancer treatment, berberine's ability to activate RXRα is still inferior, but its better safety and selectivity provide a basis for this invention. Through rational design and modification targeting RXRα directly with berberine, a total synthetic strategy is used to perform "site-directed mutagenesis" on berberine to achieve the addition or knockout of some functional groups, synthesizing a new dehydroberberine derivative, DH-Ber-12. This study analyzed the effects of DH-Ber-12 on RXRα transcriptional activation activity; tested the effects of the derivative on the proliferation of human colon cancer cells using the MTT assay and Edu assay; examined the effects of the derivative on the Wnt signaling pathway using Western blotting and mammalian two-hybrid systems; detected the effects of the derivative on the cell cycle of colon cancer cells using Western blotting; assessed the inhibitory effects of the derivative on tumors using nude mouse tumorigenesis assays, Western blotting, and immunohistochemistry; detected the binding affinity and structure-activity relationship of the derivative to RXRα using fluorescence titration and circular dichroism spectroscopy; and determined the bioavailability of DH-Ber-12 using pharmacokinetic analysis. The results demonstrated that DH-Ber-12 was superior to berberine in all four aspects: RXRα transcriptional activation activity, solubility, antitumor activity, and bioavailability. Attached Figure Description

[0037] Figure 1 This describes the synthetic route for the dehydroberberine derivative DH-Ber-12.

[0038] Figure 2 The image shows the 1H NMR spectrum of DH-Ber-12.

[0039] Figure 3 This is the DH-Ber-12 13C NMR spectrum.

[0040] Figure 4 The effects of Ber, Ber-12, and DH-Ber-12 on RXRα transcriptional activation activity in HCT116 colon cancer cells. Figure 4 In human colon cancer cells HCT116, after co-transfection with pBind-GAL4-RXRα / LBD and the internal control pG5Luc reporter gene plasmid for 24 h, the cells were incubated with berberine and NBD-125 for 15 h, respectively. Finally, dual-luciferase reporter gene expression analysis was performed. Data for each group were collected.

[0041] It is expressed as Mean±SEM.

[0042] Figure 5 The inhibitory effects of Ber, Ber-12, and DH-Ber-12 on the proliferation of HCT116 colon cancer cells were investigated (MTT assay). Figure 5 In human colon cancer cells HCT116, the proliferation rate of HCT116 colon cancer cells was detected by MTT assay after treatment with 5.0 μM, 10.0 μM, 20.0 μM, 40.0 μM, 80.0 μM and 100 μM of Ber, Ber-12 and DH-Ber-12 for 15 h.

[0043] Figure 6 The inhibitory effects of Ber, Ber-12, and DH-Ber-12 on the proliferation of HCT116 colon cancer cells were investigated (EdU assay). Figure 6 In human colon cancer cells HCT116, the proliferation rate of HCT116 colon cancer cells was detected by EdU method after treatment with 5.0 μM, 10.0 μM, 20.0 μM, 40.0 μM and 80.0 μM of Ber, Ber-12 and DH-Ber-12 for 15 h.

[0044] Figure 7 Ber, Ber-12, and DH-Ber-12 inhibited the expression of β-catenin protein in HCT116 colon cancer cells. Figure 7 In human colon cancer cells HCT116, after treatment with 25 μM, 50 μM Ber, Ber-12 and DH-Ber-12 for 15 h, respectively, the expression of β-catenin protein in colon cancer cells HCT116 was detected by immunoblotting.

[0045] Figure 8 The effects of Ber, Ber-12, and DH-Ber-12 on cyclins in HCT116 colon cancer cells. Figure 8In human colon cancer cells HCT116, after treatment with 20 μM, 40 μM and 80 μM Ber, Ber-12 and DH-Ber-12 for 15 h, respectively, the expression of cell cycle proteins c-Myc, CDK1, Cyclin B1 and p21WAF1 / CIP1 in colon cancer cells HCT116 was detected by Western blotting.

[0046] Figure 9 The effects of Ber, Ber-12, and DH-Ber-12 on xenograft growth in nude mice. Figure 9 In a study conducted under sterile conditions, BALB / c nude mice were subcutaneously injected with HCT116 colon cancer cells. Nude mouse xenograft experiments were performed to examine the effects of Ber, Ber-12, and DH-Ber-12 on the growth of HCT116 human colon cancer cell xenografts.

[0047] Figure 10 The fluorescence spectra of Ber, Ber-12, and DH-Ber-12 are shown.

[0048] Figure 11 The UV-Vis absorption spectra of Ber, Ber-12, and DH-Ber-12 are shown.

[0049] Figure 12 The effects of Ber, Ber-12, and DH-Ber-12 on the proliferation of normal human intestinal epithelial cells NCM460 were investigated. Figure 12 In human normal epithelial cells NCM460, after treatment with 5.0 μM, 10.0 μM, 20.0 μM, 40.0 μM, 80.0 μM and 100.0 μM of Ber, Ber-12 and DH-Ber-12 for 15 h, the proliferation rate of normal epithelial cells NCM460 was detected by MTT assay. Detailed Implementation

[0050] Example 1

[0051] Preparation of Compound 4: In a reaction flask, 702 mg (3.8 mmol) of Compound 3 was dissolved in 50 mL of dioxane until fully dissolved to prepare solution A. 583 mg (5.3 mmol) of selenium dioxide was dissolved in 50 mL of 1,4-dioxane until fully dissolved to prepare solution B. Solution A was then added dropwise to solution B over 30 minutes, and the mixture was refluxed at 120 °C for 8 hours. The reaction was monitored by TLC. After completion, the mixture was filtered, and the filtrate was concentrated under vacuum. The residue was dispersed in water, and the pH was adjusted to 9 by adding 1 M sodium hydroxide aqueous solution. The solution was then extracted with diethyl ether (100 mL × 4), and the organic layer was dried over sodium sulfate. After removing the solvent under vacuum, the residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 4:1) to give compound 4 (522 mg) as a white solid, in 70% yield.

[0052]

[0053] Example 2

[0054] Preparation of compound 5: In a reaction flask, 150 mg (0.75 mmol) of compound 4 was dispersed in 3 mL of anhydrous ethanol, and then 78.4 mg (1.12 mmol) of hydroxylamine hydrochloride was added. The mixture was magnetically stirred and refluxed at 160 °C for 2 hours. After cooling to room temperature, the resulting yellowish-white precipitate was collected by filtration and dried under vacuum to give a white solid 5 (139 mg), with a yield of 85%.

[0055]

[0056] Example 3

[0057] Preparation of Compound 6: 100 mg (0.462 mmol) of Compound 5 and 107 mg (0.462 mmol) of 2-methoxybenzyl bromide were dissolved in 2 mL of dry N,N-dimethylformamide (DMF). The mixture was magnetically stirred at 100 °C for 1 hour. The oil bath was then removed, and the solution was stirred at room temperature for another 48 hours. 20 mL of dry diethyl ether (Et₂O) and 10 mL of ethyl acetate (EtOAc) were added to the solution, resulting in a yellow precipitate. The precipitate was filtered and washed twice with Et₂O. After drying, the precipitate was dispersed in 2 mL of concentrated hydrochloric acid, and the mixture was stirred at 100 °C for 30 minutes. After cooling to room temperature, an orange precipitate formed. The precipitate was filtered, washed twice with diethyl ether, and dried to give an orange solid (44 mg), yield 30%.

[0058]

[0059] The berberine derivative DH-Ber-12 described in this invention can be used in the preparation of antitumor drugs. These antitumor drugs include those for colon cancer and rectal cancer.

[0060] The present invention modifies berberine to obtain a berberine derivative DH-Ber-12 (compound DH-Ber-12), which is superior to berberine in terms of activity, solubility, and inhibitory activity against colon cancer cells.

[0061] Example 4

[0062] Solubility assessment of berberine and DH-Ber-12: The newly synthesized berberine derivative DH-Ber-12 was observed and compared with berberine. A summary table of the relevant physical properties of Ber, Ber-12, and DH-Ber-12 is shown in Table 1. The three share some similarities in physical properties, but also have differences. First, structurally, all three have the same overall architecture, possessing an isoquinoline dihydroisoquinoline tetracyclic structure, with one quaternary ammonium salt unit and two alkoxy-substituted benzene rings within the molecule. The difference lies in the following: berberine has a methylenedioxy group at C2 and C3, and a separate methoxy group at C9 and C10; Ber-12 retains only one methoxy group at C3 and C9 respectively; and DH-Ber-12 not only retains one methoxy group at C3 and C9 respectively, but also loses one and two hydrogens, forming a double bond. Second, all three are yellow in both solid and solution form. Berberine is slightly darker than Ber-12, but DH-Ber-12 is the darkest. Third, regarding solubility, in DMSO solvent, the complete dissolution time of Ber-12 is one-tenth that of berberine, and the complete dissolution time of DH-Ber-12 is half that of Ber-12. Conservatively, the solubility of DH-Ber-12 (DMSO) can be considered to be increased by 20 times. When using sterile double-distilled water as the solvent, the complete dissolution time of Ber-12 is one-sixth that of berberine, and the time required for the complete dissolution of DH-Ber-12 is only one-third that of Ber-12. Therefore, the solubility of DH-Ber-12 (water) can be considered to be increased by 10 times. Thus, the modified berberine derivative DH-Ber-12 has at least 8 times higher solubility than berberine, making it a compound with better solubility.

[0063] Table 1

[0064]

[0065] Example 5

[0066] Effects of Ber, Ber-12, and DH-Ber-12 on the proliferation of HCT116 colon cancer cells: The MTT assay was used to examine the inhibitory effects of berberine, Ber-12, and DH-Ber-12 on the proliferation of HCT116 colon cancer cells. HCT116 colon cancer cells were cultured in MEM complete medium (Gibco, 61100061, supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM vitamins, 100 U / mL penicillin, 100 U / mL streptomycin, and 1.5 g NaHCO3 / L) at 37°C, 5% CO2, and 95% saturated humidity in a CO2 incubator. After the cells reached near-confluence, they were digested with 0.25% trypsin, and the cell concentration was adjusted to 102. 4 / mL, into a 96-well cell culture plate, with 10 cells per well. 4 The cells were cultured overnight. After cell attachment, the original culture medium was discarded. Cells were then grouped according to the type and concentration of the drug used for treatment: drug-free control group, solvent (DMSO) control group, berberine group (5, 10, 20, 40, 80, 120 μM), Ber-12 group (5, 10, 20, 40, 80, 120 μM), and DH-Ber-12 group (5, 10, 20, 40, 80, 120 μM). The final volume of cell culture medium in each well was 200 μL. Each group was divided into 5 replicates, and cultured for another 15 h. Next, the cells were washed twice with pH 7.4 PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄). 20 μL of 5 mg / mL MTT and 180 μL of fresh MEM complete culture medium were added to each well, and the cells were cultured for another 4 h. The supernatant was then discarded. Add 200 μL of DMSO to each well and shake at 37°C for 10 min to completely dissolve the blue-purple formazan crystals. Immediately measure the absorbance at 490 nm using a Bio-Rad Model 680 microplate reader, with 630 nm as the reference wavelength. Use the same passaged cells for each experiment. Calculate the inhibition rate using the following formula: Inhibition rate (%) = (OD value of control group - OD value of experimental group) / OD value of control group × 100%.

[0067] The results are as follows Figure 5 As shown, Ber, Ber-12, and DH-Ber-12 all exhibited inhibitory effects on the proliferation of HCT116 colon cancer cells in a concentration-dependent manner; the higher the drug concentration, the stronger the inhibitory effect. Furthermore, a parallel comparison of the tumor-inhibiting effects of Ber, Ber-12, and DH-Ber-12 at the same concentration revealed that, at concentrations of 5, 10, 20, 40, 80, and 120 μM, DH-Ber-12 showed significantly superior inhibitory effects on the proliferation of colon cancer cells compared to berberine.

[0068] Example 6

[0069] EdU reagent kit assay: The EdU reagent kit assay was used to investigate the inhibitory effects of Ber, Ber-12 and DH-Ber-12 on the proliferation of HCT116 colon cancer cells.

[0070] (1) Cell culture: Logarithmically growing colon cancer HCT116 cells were cultured at 100 cells per well. 5 Each seed was inoculated into a 96-well plate and cultured until it reached the normal growth stage.

[0071] (2) Drug treatment: After cell adhesion, the original culture medium was discarded. Cells were grouped according to the type and concentration of the drug used for treatment: drug-free control group, solvent (DMSO) control group, Ber group (5, 10, 20, 40, 80 μM), Ber-12 group (5, 10, 20, 40, 80 μM), and DH-Ber-12 group (5, 10, 20, 40, 80 μM). The final volume of cell culture medium in each well was 200 μL. Each group was replicated in 5 places and cultured for 12 h.

[0072] (3) EdU labeling: Dilute the EdU solution with MEM complete medium to prepare an appropriate amount of 50 μM EdU medium; add 100 μL of 50 μM EdU medium to each well and incubate for 2 h, then discard the medium.

[0073] (4) Cell fixation: Add 100 μL of cell fixation solution (PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min; add 2 mg / mL glycine to each well, decolorize and incubate on a shaker for 5 min, then discard the glycine solution; add 100 μL of PBS to each well, decolorize and wash on a shaker for 5 min, then discard the PBS; add 100 μL of permeabilizer (PBS containing 0.5% Tritium X-100) to each well, decolorize and incubate on a shaker for 10 min; wash once with PBS for 5 min.

[0074] (5) Apollo staining: Add 100 μL of 1*Apollo staining reaction solution to each well, incubate in the dark at room temperature on a decolorizing shaker for 30 min, then discard the staining reaction solution; add 100 μL of penetrant to decolorize and wash on a shaker 2-3 times, 10 min each time, then discard the penetrant; add 100 μL of methanol to each well and wash twice, 5 min each time; wash once with PBS, 5 min each time.

[0075] (6) DNA staining: Dilute reagent F with deionized water at a ratio of 100:1 to prepare an appropriate amount of 1*Hoechst33342 reaction solution and store it in the dark; add 100 μL of 1*Hoechst33342 reaction solution to each well, incubate in the dark, at room temperature, and on a decolorizing shaker for 30 min, then discard the staining reaction solution; wash each well 3 times with 100 μL of PBS each time.

[0076] (7) Image acquisition and analysis: Immediately after staining, observation and statistical analysis were performed under a microscope. Results are as follows: Figure 7 As shown, Ber, Ber-12, and DH-Ber-12 all exhibited inhibitory effects on the proliferation of HCT116 colon cancer cells in a concentration-dependent manner; the higher the drug concentration, the stronger the inhibitory effect. Furthermore, a parallel comparison of the tumor-inhibiting effects of Ber, Ber-12, and DH-Ber-12 at the same concentration revealed that, at concentrations of 5, 10, 20, 40, and 80 μM, DH-Ber-12 showed significantly superior inhibitory effects on the proliferation of colon cancer cells compared to berberine.

[0077] Implementation Case 7

[0078] Effects of Ber, Ber-12, and DH-Ber-12 on β-catenin protein expression in HCT116 colon cancer cells: HCT116 colon cancer cells were cultured in MEM complete medium (Gibco, 61100061, supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM vitamins, 100 U / mL penicillin, 100 U / mL streptomycin, and 1.5 g NaHCO3 / L) at 37°C, 5% CO2, and 95% saturated humidity in a CO2 incubator. After cells reached near-confluence, they were digested with 0.25% trypsin, and the cell concentration was adjusted to 1 × 10⁶ cells / mL. 7 Add cells / mL to 6-well cell culture plates, with 2 × 10⁶ cells per well. 5The cells were cultured overnight. After cell attachment, the original culture medium was discarded, and the cells were grouped according to the type and concentration of the drug used to treat them: drug-free control group, solvent (DMSO) control group, Ber group (20, 40, 80 μM), Ber-12 group (20, 40, 80 μM), and DH-Ber-12 group (20, 40, 80 μM). The final volume of cell culture medium in each well was 2 mL. Each group was divided into 3 replicates, and the cells were cultured for another 12 h. Then, the cells were washed twice with PBS buffer (pH 7.4) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), and 200 μL of lysis buffer was added to each well. The cells were sonicated on ice, centrifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was collected to determine the protein concentration.

[0079] Protein concentration determination: The Thermo Biotech BCA protein concentration assay kit was used. The experimental principle is that proteins can convert Cu under alkaline conditions. 2+ Transformation into Cu + BCA can react with Cu + The protein binds to form a purple product with a strong absorbance at 562 nm. This absorbance shows a linear relationship with protein concentration in the range of 20–2000 μg / ml. The specific experimental steps are as follows:

[0080] (1) Add 8 μL of physiological saline to each well, and then add 2 μL of the protein sample to be tested; dilute the standard with 2 mg / mL BSA according to the serial dilution method;

[0081] (2) Mix Reagent A and Reagent B in a ratio of 50:1 to prepare the total required working solution. Prepare the solution immediately before use.

[0082] (3) Add 200 μL of working solution to each well, gently tap for 30 seconds to mix thoroughly;

[0083] (4) Cover the 96-well plate with the lid and place at 37°C for 25 min;

[0084] (5) Place the sample in the microplate reader and set the absorbance value to 570 nm for measurement;

[0085] (6) Derive the corresponding formula based on the concentration of the standard, and then calculate the concentration of the sample to be tested and the volume of the sample loaded.

[0086] Implementation Case 8

[0087] Protein electrophoresis: Take 30 μg of protein sample, add an equal volume of 2×SDS loading buffer, boil at 95℃ for 5 min, and electrophores at a constant current of 60 mA in a discontinuous SDS-PAGE gel.

[0088] Implementation Case 8

[0089] Electrotransfer: The electrotransfer solution was pre-cooled at 4℃. After cutting the gel, a PVDF membrane (pretreated with methanol) slightly larger than the gel and filter paper were immersed in the electrotransfer solution. After the PVDF membrane was attached to the gel, filter paper was placed on both sides to remove air bubbles. The membrane was then placed in the electrotransfer cell with the positive electrode facing the cell and electrotransferred at -20℃ (100 V, 60 min).

[0090] Implementation Case 9

[0091] Antigen-antibody reaction: Blocking buffer was used to cover the membrane, and the membrane was incubated at room temperature for 1 hour. The blocked membrane was then incubated with the corresponding primary antibody at room temperature for 3 hours. The membrane was washed three times with TBST for 5 minutes each time. Then the corresponding secondary antibody was added, and the membrane was incubated at room temperature for 2 hours.

[0092] Implementation Case 10

[0093] ECL assay: Wash 3 times with TBST, 10 min each time. Before use, mix ECL solution A and solution B at a ratio of 1:1 (V / V), drop the mixture onto the membrane surface in a dark room, incubate for 1 min, and then expose the membrane with film.

[0094] Implementation Case 11

[0095] Lysis Buffer: 50mM HEPES (pH 7.4), 100mM NaCl, 10% glycerol, 1% Triton-X100, 1.5mM MgCl2, 25mM NaF, add 1mM PMSF just before use.

[0096] 10× Lower gel buffer (1L): 422.5g Tris, 10g SDS, pH adjusted to 8.8 with HCl.

[0097] 4× Top gel buffer (1L): 60.6g Tris, 4g SDS, pH adjusted to 6.8 with HCl.

[0098] 10% lower gel (10 mL): 3.33 mL 30% acrylamide-methylenebisacrylamide (29:1), 1 mL 10× lower gel buffer, 5.6 mL water, 100 μL 10% ammonium persulfate, 10 μL TEMED.

[0099] 4% Top Gel (5 mL): 0.67 mL 30% Acrylamide-N-methylbisacrylamide (29:1), 1.25 mL 4× Top Gel Buffer, 3.05 mL Water, 50 μL 10% Ammonium Persulfate, 5 L TEMED.

[0100] 2×SDS loading buffer (10mL): 2mL glycerol, 4mL 10% SDS, 2mL 4× top gel buffer, 1mL β-mercaptoethanol, 0.002g bromophenol blue.

[0101] 10× Electrophoresis Buffer (1L): 30g Tris, 144g Glycine, 10g SDS.

[0102] 10× Electroporation Buffer (1L): 24.25g Tris, 112.5g Glycine, add methanol to a final concentration of 10% just before use.

[0103] 10×TBS(1L): 24.2g Tris, 80g NaCl, pH adjusted to 7.6 with HCl.

[0104] TBST elution buffer (1L): 100mL 10×TBS, 1mL Tween-20.

[0105] Blocking solution: 5% skim milk powder dissolved in TBST.

[0106] Primary antibody dilution buffer: 5% BSA, 0.03% NaN3 dissolved in TBST.

[0107] The results are as follows Figure 8 As shown, berberine, Ber-12, and DH-Ber-12 all inhibited the protein expression of β-catenin in HCT116 colon cancer cells in a concentration-dependent manner; the higher the drug concentration, the stronger the inhibitory effect. Furthermore, a parallel comparison of the tumor-inhibiting effects of Ber, Ber-12, and DH-Ber-12 at the same concentration revealed that, at concentrations of 20, 40, and 80 μM, DH-Ber-12 exhibited superior inhibitory effects on β-catenin protein expression compared to Ber-12 and Ber.

[0108] Implementation Case 12

[0109] Tumor formation experiment in nude mice: Nude mice were housed at the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine. After reaching 6-7 weeks of age and weighing 18-20g, tumor transplantation experiments were conducted. Colon cancer cells (HCT116) were cultured in MEM complete medium (Gibco, 61100061, supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, 1mM vitamins, 100 U / mL penicillin, 100 U / mL streptomycin, and 1.5 g NaHCO3 / L) at 37℃, 5% CO2, and 95% saturated humidity in a CO2 incubator. Once the cells reached near-confluence, they were digested with 0.25% trypsin, and the cell concentration was adjusted to 1×10⁶ cells / mL. 7 0.1 mL / mL was injected subcutaneously into the right back of nude mice (1×10⁻⁶). 6 (cells). After palpable xenografts formed in mice, they were randomly divided into 4 groups of 8 mice each. One group was administered physiological saline (5 μL / g body weight) by gavage, while the other three groups were administered berberine (100 mg / kg body weight, dissolved in 0.5% CMC-Na solution), Ber-12 (100 mg / kg body weight, dissolved in 0.5% CMC-Na solution), and DH-Ber-12 (100 mg / kg body weight, dissolved in 0.5% CMC-Na solution), respectively, by gavage once daily. After 15 days, the mice were euthanized by cervical dislocation, the xenografts were removed, photographed, and weighed.

[0110] Implementation Case 13

[0111] Fluorescence spectroscopy detection: Figure 10 The figure shows the photoluminescence fluorescence spectra of Ber, Ber-12, and DH-Ber-12. The figure shows that the fluorescence emission wavelength of DH-Ber-12 (519 nm) is significantly longer than that of Ber-12 (413 nm) and Ber (402 nm), indicating that the performance of DH-Ber-12 has been improved.

[0112] Implementation Case 14

[0113] UV-Vis absorption: Figure 11 The figures show the UV-Vis absorption spectra of Ber, Ber-12, and DH-Ber-12. The UV-Vis absorption of DH-Ber-12 is significantly red-shifted compared to Ber-12 and Ber, indicating that the microscopic electronic energy levels of DH-Ber-12 have changed, reducing the effective bandgap and thus improving its performance.

[0114] Implementation Case 15

[0115] Tumor selectivity of Ber, Ber-12, and DH-Ber-12: The specific implementation method is the same as in Example 2-1. The MTT assay was used to examine the effects of Ber, Ber-12, and DH-Ber-12 on the proliferation of HCT116 colon cancer cells and NCM460 colon epithelial cells. The experimental results are as follows: Figure 12 As shown, Ber, Ber-12, and DH-Ber-12 all exhibited inhibitory effects on the proliferation of HCT116 colon cancer cells in a concentration-dependent manner, meaning that the higher the drug concentration, the stronger the inhibitory effect. Furthermore, the inhibitory effect of DH-Ber-12 was more pronounced than that of berberine, showing significantly better results at concentrations of 5, 10, 20, 40, 80, and 120 μM. In contrast, at 30 μM, Ber, Ber-12, and DH-Ber-12 had virtually no inhibitory effect on the proliferation of the colon epithelial cell line NCM460; at 50 μM, both showed slight inhibitory effects on the proliferation of NCM460 colon epithelial cell line, but none were as effective as their inhibitory effect on KM12C colon cancer cells. These results show that Ber, Ber-12, and DH-Ber-12 have minimal damaging effects on normal colonic epithelial cells, indicating that the modified derivative DH-Ber-12 retains the advantages of berberine itself, such as low toxicity and high selectivity. This allows the drug to exert its anti-cancer effects while minimizing damage to normal colonic cells. Furthermore, this suggests that future development and application of DH-Ber-12 could involve clinical trials within a specific concentration range to reduce clinical toxicity and improve efficacy.

[0116] Implementation Case 16

[0117] Pharmacokinetic assays were performed to determine the bioavailability of Ber, Ber-12, and DH-Ber-12. Eighteen SPF-grade male SD rats, aged 6–8 weeks, were purchased from the Animal Experiment Center of Jiangxi University of Traditional Chinese Medicine. After one week of acclimatization, the rats were randomly divided into six groups: an intravenous injection group (IV group) and a gavage group (PO group), with three rats in each group. The IV group received a dose of 5 mg / kg, and the PO group received a dose of 100 mg / kg. At 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 36 hours after drug administration, 200 μL of blood was collected from the tail vein, anticoagulated with heparin sodium, centrifuged at 2000 rpm and 4℃ for 10 min, and 100 μL of the supernatant plasma was accurately measured and placed in another EP tube, which was stored at -60℃ for later use. Take 50 μL of plasma and add it to a 1.5 mL tube. Add 200 μL of acetonitrile containing the internal standard protonated to precipitate proteins. Swirl the mixture, centrifuge at 14000 rpm for 5 min, and inject 200 μL of the supernatant for LC-MS / MS (UPLC (Shimadzu) chromatography system) analysis.

[0118] Chromatographic separation was performed using a C18 column (50 × 2.1 mm, 1.7 μm), with the column temperature maintained at 35 °C, column pressure maintained at 25 MPa, and flow rate maintained at 1.0 mL / min. The following mobile phase was used:

[0119] A: Aqueous phase (0.1% phosphoric acid aqueous solution containing 10 mg ammonium acetate)

[0120] B: Organic phase (acetonitrile solution)

[0121] The mobile phase conditions for UPLC are shown in Table 2.

[0122] Table 2

[0123]

[0124] The bioavailability results of berberine, Ber-12, and DH-Ber-12 are shown in Table 3.

[0125] Table 3

[0126]

[0127] The main pharmacokinetic parameters of Ber, Ber-12, and DH-Ber-12 showed that, compared with berberine and Ber-12, DH-Ber-12 had a longer terminal elimination half-life (Ti). 1 / 2 The peak concentration (C) was shortened by 70%, and the peak drug concentration (C) was reduced. max The drug efficacy increased by 27.31 times, the area under the curve (AUC) increased by 27.28 times, the mean residence time (MRT) decreased by 60%, and the bioavailability (F%) increased by 39 times.

Claims

1. Preparation and application of novel (3,10-dimethoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline-7-hydrochloride)-dehydrocorydaline derivative DH-Ber-12, characterized in that: (1) 3 (6-methoxy-3-methylisoquinoline, which can be directly purchased) is used as a starting material, a selenium dioxide catalyst is added in an inert atmosphere, and the intermediate compound 4 is prepared by refluxing in a 1,4-dioxane solution at a certain temperature for several hours; (2) compound 4 is dispersed in ethanol, and a certain amount of hydroxylamine hydrochloride is added, and compound 5 is prepared by refluxing at a certain temperature for several hours; (3) compound 5 and 2-methoxybenzyl bromide are dissolved in N,N-dimethylformamide in a certain proportion, stirred and reacted at a certain temperature for several hours, then cooled to room temperature and stirred for several hours, and then a certain amount of diethyl ether and ethyl acetate is added to prepare derivative 6; (3,10-dimethoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline-7-chloride)-dehydrocorydaline derivative 6 (i.e. general formula I). In step one, compound 3 is used as a starting material, which is dissolved in dioxane, and selenium dioxide is dissolved in dioxane; the solution of compound 3 is added dropwise to the selenium dioxide solution, and magnetic stirring and reflux are continued at a certain temperature, and thin layer chromatography (TLC) is monitored; after the raw material is completely reacted, the obtained filtrate is concentrated under vacuum, then extracted with diethyl ether for 3-5 times, and the organic layer is dried with sodium sulfate. After removing the solvent by vacuum concentration, the compound 4 is obtained by rapid silica gel chromatography separation and purification, concentration, and drying. In step one, the concentration of compound 3 in dioxane is 0.05-0.1 mmol / mL, the concentration of selenium dioxide in dioxane is 0.05-0.15 mmol / mL, the reflux reaction temperature of the mixture is 100-130℃, the reflux time is 6-10 hours, and petroleum ether and ethyl acetate are used as eluents for rapid silica gel chromatography separation and purification, with petroleum ether: ethyl acetate = 5:1-3:

1. In step two, compound 4 is used as a starting material, which is first dispersed in ethanol, then hydroxylamine hydrochloride is added, and magnetic stirring and reflux are continued, then the mixture is cooled to room temperature, concentrated under vacuum, and dried to obtain compound 5.

2. Preparation of novel (3,10-dimethoxy-12,13-dihydroisoquinoline[3,2- a]isoquinoline-7-hydrochloride)-dehydrocorydaline derivatives and their use, according to claim 1, characterized by the fact that: In step two, the concentration of compound 4 dispersed in ethanol is 0.15-0.35 mmol / mL, the amount of hydroxylamine hydrochloride is 1-2 times that of compound 4, and the reaction temperature is maintained at 130-170℃, and the reflux time is 1-3 hours.

3. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-hydrochloride) - dehydrocorydaline derivatives and their use, according to claim 1, characterized by the fact that: In step three, compound 5 is used as a starting material, which is dissolved in dry N,N-dimethylformamide together with 2-methoxybenzyl bromide, and magnetic stirring is continued at room temperature after a certain period of reaction; after complete reaction, diethyl ether and ethyl acetate are added, then filtered, washed with diethyl ether for 2-3 times, dried, and then the precipitate is dispersed in concentrated hydrochloric acid, magnetic stirring is continued, the mixture is cooled to room temperature, washed with diethyl ether for 2-3 times, dried, and concentrated to obtain compound 6.

4. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-hydrochloride) - dehydrocorydaline derivatives and their use, according to claim 1, characterized by the fact that: ​ 5. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-chloride) - dehydrocorydaline derivatives and their use according to claim 1, characterized by that: ​ 6. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-hydrochloride) - dehydrocorydaline derivatives and their use, according to claim 1, characterized by that: ​ 7. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-hydrochloride) - dehydrocorydaline derivatives and their use, according to claim 1, characterized by that: ​ 8. Preparation of novel (3, 10-dimethoxy-12, 13-dihydroisoquinoline [3, 2-a] isoquinoline-7-hydrochloride) - dehydrocorydaline derivatives and their use, according to claim 1, characterized by the fact that: In step three, the molar ratio of compound 5 to 2-methoxybenzyl bromide is 0.5-1.5, the amount of N,N-dimethylformamide is 0.2-0.3 mmol / mL, the reaction temperature is 90-110°C, the reaction time is 0.5-1.5 hours, the stirring time at room temperature is 45-51 hours, the amount of ether added is 8-12 times of N,N-dimethylformamide, the amount of ethyl acetate added is 3-8 times, the amount of concentrated hydrochloric acid is 0.8-1.2 times of N,N-dimethylformamide, the reaction temperature of the precipitate dispersed in concentrated hydrochloric acid is 80-120°C, and the reaction time is 20-40 minutes.

9. Preparation and use of novel (3,10-dimethoxy-12,13-dihydroisoquinoline[3,2- a]isoquinoline-7-hydrochloride)-dehydrocorydaline derivatives according to claims 1 and 2, characterized by the fact that: The novel (3,10-dimethoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline-7-hydrochloride) dehydrogenated berberine derivative is: (1) 3,10-dimethoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 7), the structural formula is: (2) 3-fluoro,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 8), the structural formula is: (3) 3-difluoromethyl,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 9), the structural formula is: (4) 3-trifluoromethyl,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 10), the structural formula is: (5) 3-difluoromethoxy,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 11), the structural formula is: (6) 3-trifluoromethoxy,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 12), the structural formula is: (7) 3-methoxy,10-fluoro-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 13), the structural formula is: (8) 3-methoxy,10-difluoromethyl-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 14), the structural formula is: (9) 3-methoxy,10-trifluoromethyl-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 15), the structural formula is: (10) 3-methoxy,10-difluoromethoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 16), the structural formula is: (11) 3-difluoromethoxy,10-methoxy-12,13-dihydroisoquinoline[3,2-a]isoquinoline hydrochloride (compound 17), the structural formula is:

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