CN104208061A - Medical application of berberine derivative - Google Patents

Abstract

The present invention provides the application of a compound of formula (I) or a salt thereof in the preparation of a drug for treating malignant glioma or a mitochondrial targeted drug delivery system: Formula (I), wherein, R is C ₁₀ -C ₁₈ alkyl, or R is benzyl. The present invention finds through experiments that, compared with the control, the berberine derivatives of the present invention can not only more effectively inhibit the proliferation of glioma cells, but also effectively inhibit the migration and invasion abilities of cells. In addition, these compounds localize well to mitochondria and thus serve as mitochondria-targeted drug delivery systems.

Description

Medicinal uses of berberine derivatives

technical field

The present invention relates to the new application of berberine derivatives, especially the new application of C9-O substituted long-chain alkyl or benzyl derivatives in treating malignant glioma.

Background technique

Malignant glioma (Malignant Glioma) is the most common primary malignant tumor in the central nervous system, accounting for 46% of intracranial tumors. Surgery is the preferred treatment method, but because of its invasive growth and no obvious boundary with normal brain tissue, surgery is difficult to remove, and the postoperative recurrence rate is as high as 96%. Even with supplementary radiotherapy and chemotherapy, the average postoperative survival period is no more than 14 months, and the prognosis is extremely poor. The incurability of malignant glioma is closely related to its strong ability of migration and invasion. Therefore, inhibiting the migration and invasion of glioma cells is extremely critical for the treatment of malignant gliomas. However, the existing treatment methods cannot effectively inhibit the migration and invasion of malignant glioma, resulting in poor therapeutic effect, and there are many defects such as neurotoxicity, hematological toxicity, and drug resistance.

In addition, the pathological process of various diseases, including tumors, is associated with mitochondrial damage. There are large differences in the structure and function of mitochondria between tumor cells and normal cells. The abnormalities of intracellular biochemical events directly related to mitochondria, such as changes in glucose metabolism, ATP generation disorders, calcium ion accumulation, oxidative stress, etc. The degree of dysfunction is an important reason for the development of tumors. Meanwhile, mtDNA mutations and excess ROS in tumor cells have also been reported to promote tumor cell metastasis. This makes it possible to target drugs to the mitochondria of tumor cells and selectively act on tumor cells through mitochondria, which is of great significance for the targeted treatment of highly aggressive tumors such as malignant glioma.

Berberine is an isoquinoline alkaloid extracted and isolated from Coptis chinensis and other traditional Chinese medicinal materials. It is safe to take orally and has no blood, cardiovascular, liver and kidney toxicity if taken in large quantities for a long time. Studies have shown that berberine can inhibit the migration and invasion of liver cancer, human tongue squamous cell carcinoma, melanoma, breast cancer, bladder cancer, etc., but there is no report on the impact on glioma migration and invasion. On the other hand, although berberine has inhibitory activity against certain tumors, its efficacy is not ideal.

The invention intends to develop a safe, effective and low-toxic chemotherapy drug, which can effectively inhibit the migration and invasion of malignant glioma cells.

Contents of the invention

In order to solve the above problems, the present invention provides a new treatment scheme for malignant glioma, which is realized based on the unexpected inhibitory effect of berberine derivatives on malignant glioma cells.

According to the first aspect of the present invention, the present invention provides the use of a compound of formula (I) or a salt thereof in the preparation of a drug for treating malignant glioma:

,

Wherein, R is C ₁₀ -C ₁₈ alkyl, or R is benzyl. The C ₁₀ -C ₁₈ alkyl group includes C ₁₀ -C ₁₈ n-alkyl group or their isomers.

In a preferred embodiment, R is a C ₁₀ -C ₁₈ n-alkyl group, such as n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-decyl Pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl. More preferred is a C ₁₀ -C ₁₆ n-alkyl group. More preferably, R is a C ₁₂ -C ₁₆ n-alkyl group. More preferably, R is n-dodecyl or n-hexadecyl.

In a preferred embodiment, the salt is selected from hydrobromide, hydroiodide, hydrofluoride, hydrochloride, sulfate, nitrate, phosphate, citrate, acetate and lactic acid Salt, preferably hydrobromide, hydrochloride or sulfate.

According to a preferred embodiment of the present invention, the compound is selected from: 9-O-n-decyl berberine, 9-O-n-dodecyl berberine, 9-O-n-hexadecyl berberine base, 9-O-n-octadecylberberine, and 9-O-benzylberberine.

According to one embodiment of the present invention, the medicament comprises at least one additional ingredient effective for the treatment of malignant glioma for use in combination with the derivative of the present invention. According to one embodiment of the present invention, the medicament contains pharmaceutical excipients to prepare the medicament into a suitable dosage form.

The inventors found that 9-O substituted long-chain alkyl or benzyl berberine derivatives are unexpectedly better than other berberine derivatives and Berberine itself. The present invention finds through experiments that, compared with the control, the berberine derivatives can not only more effectively inhibit the proliferation of glioma C6 cells, but also effectively inhibit the migration and invasion abilities of the cells. The possible reason is that when the lipophilic group (long-chain alkyl or benzyl) is combined with the parent drug berberine by ether linkage, the fat solubility of the compound is improved, and the compound is effectively permeated by improving transmembrane transport. Through the biofilm, it is transported to the lesion site and cells, thereby enhancing the efficacy of the drug.

According to the second aspect of the present invention, the present invention provides the use of a compound of formula (I) or a salt thereof in the preparation of a mitochondria-targeted drug delivery system:

,

R is C ₁₀ -C ₁₈ alkyl or benzyl. The C ₁₀ -C ₁₈ alkyl group includes C ₁₀ -C ₁₈ n-alkyl group or their isomers.

In a preferred embodiment, R is a C ₁₀ -C ₁₈ n-alkyl group, such as n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-decyl Pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl. More preferred is a C ₁₀ -C ₁₆ n-alkyl group. More preferably, R is a C ₁₂ -C ₁₆ n-alkyl group. More preferably, R is n-dodecyl or n-hexadecyl.

According to a preferred embodiment of the present invention, the compound is selected from: 9-O-decyl berberine, 9-O-dodecyl berberine, 9-O-hexadecyl berberine, 9 -O-octadecylberberine, and 9-O-benzylberberine.

In a preferred embodiment, the salt is selected from hydrobromide, hydroiodide, hydrofluoride, hydrochloride, sulfate, nitrate, phosphate, citrate, acetate and lactic acid Salt, preferably hydrobromide, hydrochloride or sulfate.

The mitochondrial-targeted drug delivery system comprises at least one additional pharmaceutical ingredient, which generally has difficulty entering the mitochondria by itself and does not adversely interact with the derivative of the present invention. Under the action of the derivative of the present invention, the additional pharmaceutical ingredient can be located in the mitochondria, thereby exerting its effect.

According to the third aspect of the present invention, the present invention provides a method for treating malignant glioma, the method comprising administering a therapeutically effective amount of a compound of formula (I) or a salt thereof to a patient in need thereof.

In the present invention, C ₁₀ -C ₁₈ alkyl refers to straight chain or branched chain alkyl having 10 to 18 carbon atoms, similarly, C ₁₂ -C ₁₆ alkyl refers to having 12 to 16 carbon atoms Straight chain or branched chain alkyl.

Description of drawings

Figure 1 shows the changes in cell viability after different concentrations of berberine and its derivatives acted on C6 cells for 24 h.

Figure 2 shows the changes in cell viability after different concentrations of berberine derivatives acted on C6 cells for 24 h, 48 h and 72 h, (A) derivative 9-O-dodecylberberine, referred to as B3, (B) derivative 9-O-hexadecyl berberine, referred to as B4, (C) derivative 9-O-octadecyl berberine, referred to as B5, (D) derivative 9-O-benzyl Base berberine, referred to as B6.

Figure 3 shows the results of berberine and its derivatives C6 cell scratch experiment, (A) the cell migration of the control group, berberine group and berberine derivative group after 0h and 24h of cell scratches, (B) Comparison of relative mobility between groups, compared with the control, * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001. B3 represents 9-O-dodecyl berberine; B4 represents 9-O-hexadecyl berberine; B5 represents 9-O-octadecyl berberine; B6 represents 9-O-benzyl Berberine.

Figure 4 shows the results of the Transwell migration assay of berberine and its derivatives C6 cells, (A) the cell migration of the control group, berberine group and berberine derivatives group, (B) the relative mobility among the groups Compared with the control, * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001. B3 represents 9-O-dodecyl berberine; B4 represents 9-O-hexadecyl berberine; B5 represents 9-O-octadecyl berberine; B6 represents 9-O-benzyl Berberine.

Figure 5 shows the results of the Transwell invasion assay of berberine and its derivatives C6 cells, (A) the inhibition of cell invasion by the control group, the berberine group and the berberine derivatives group; (B) the relative Mobility comparison, compared with the control, * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001. B3 represents 9-O-dodecyl berberine; B4 represents 9-O-hexadecyl berberine; B5 represents 9-O-octadecyl berberine; B6 represents 9-O-benzyl Berberine.

Fig. 6 shows confocal observation results of mitochondrial localization of berberine lipophilic derivatives in C6 (A) and U87 (B) glioma cells. Mitotracker indicates mitochondria labeled with mitotracker green, showing green fluorescence; drug indicates berberine and its lipophilic derivatives, showing yellow fluorescence; overlay indicates the superposition of the above two images. B3 represents 9-O-dodecyl berberine; B4 represents 9-O-hexadecyl berberine.

Detailed ways

Synthesis and Identification of Berberine Derivatives

Berberine hydrochloride was purchased from Xi’an Xiaocao Plant Technology Co., Ltd., with a purity of 97%; bromododecane, bromohexadecane, bromooctadecane, and benzyl bromide were all analytically pure, purchased from Aladdin Reagent Co., Ltd. Company; N, N-dimethylformamide (DMF) is analytically pure, dried with molecular sieves, and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; neutral alumina for chromatography is a product of Sinopharm Group; DMEM medium (R10 -013-cv), fetal bovine serum (FBS), trypsin, penicillin and streptomycin were all purchased from Cellgro, USA; tetramethylazozolium salt (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Company of the United States; 4% paraformaldehyde was purchased from Guangzhou Jingxin Technology Co., Ltd.; the experimental water was ultrapure water. Rat glioma C6 cells and human glioma U87 cells were from the ATCC cell bank in the United States. Millicell Cell Culture Inserts (8.0 μm PET) is a product of Millipore, USA. Avance ^Ⅲ 400MHz nuclear magnetic resonance spectrometer is a product of American Bruker; ultra-high performance liquid chromatography tandem mass spectrometer UPLC-MS/MS (TSQ Quantum Access Max) is a product of American Thermo scientific; high-speed refrigerated centrifuge (LEGEND MICRO 17R) is a product of American Thermo The product of scientific company; the microplate reader (Microplate Reader) is the product of BIO-RAD company in the United States; the inverted research microscope (IX 71) is the product of OLYMPUS company in Japan; the water-jacketed CO ₂ incubator (HEPA class100) is the product of Thermo scientific company in the United States .

An exemplary synthetic route of the berberine derivatives of the present invention is as follows:

1.1 Synthesis and Identification of Berberrubine (B2)

Intermediate B 2 was synthesized by pyrolysis method.

Take 10 g (0.0269 mol) of dried berberine hydrochloride and crack it at 190 °C for 5 h under the protection of _N2 . Cool to room temperature and wash with 50 mL of acetone to obtain 7.48 g of red crystals with a yield of 87%. ¹ H NMR (400 MHz, CD ₃ OD) δ: 9.22 (s, 1H), 7.96 (s, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.39 (s, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.02 (s, 2H), 4.52-4.62 (t, J = 6.3 Hz, 2H), 3.86 (s, 3H), 3.05-3.14 (t, J = 6.3 Hz, 2H); ¹³ C NMR (101 MHz, CD ₃ OD) Δ: 151.11, 150.85, 149.47, 147.31, 135.54, 133.74, 130.66, 122.90, 121.71, 119.63, 108.17, 105.81, 103.25, 56.79, 55.53, 28.96; ESI-MS m/z : 322 [M+H] ⁺ .

Synthesis and Identification of Berberine Derivatives B3, B4, B5, B6 and B12

Reaction of B2 with the corresponding bromide affords B3, B4, B5, B6 and B12 , respectively.

Dissolve 0.32 g (1 mmol) of B2 in 5 mL DMF, react with 4 mmol of the corresponding bromide at 80 °C for 2 h to 24 h under the protection of N ₂ , filter, collect the precipitate, load the sample by dry method, and purify on a neutral alumina column (chloroform-methanol 200-100:1), yellow crystals B3, B4 , , B5, B6 and B12 were obtained respectively. The yield is 70%~80%.

9-O-Dodecylberberine ( B3 ) ¹ H NMR (400 MHz, DMSO) δ: 9.76 (s, 1H), 8.95 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H) , 7.99 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H), 4.91 (t, J = 6.7 Hz, 2H), 4.28 (t, J = 6.7 Hz, 2H), 4.05 (s, 3H), 3.24 (t, J = 6.7 Hz, 2H), 1.81-1.94 (m, 2H), 1.42-1.52 (m, 2H), 1.39-1.22 (m , 16H), 0.85 (t, J = 6.7 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) δ: 150.34, 149.76, 147.62, 145.20, 142.84, 137.36, 133.01, 130.58, 60.612, 213, 123 , 120.16, 108.35, 105.38, 102.03, 74.22, 57.00, 55.30, 31.23, 29.43, 28.97, 28.77, 28.65, 26.30, 25.21, 22.03, 13.87 ⁺ M: ESI-MS4 m/z .

9-O-Hexadecylberberine ( B4 ) ¹ H NMR (400 MHz, DMSO) δ: 9.75 (s, 1H) 8.94 (s, 1H), 8.20 (d, J = 8.9 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.80 (s, 1H), 7.09 (s, 1H), 6.18 (s, 2H), 4.95 (t, J = 6.7 Hz, 2H), 4.29 (t, J = 6.7 Hz, 2H), 4.05 (s, 3H), 3.23 (t, J = 6.7 Hz, 2H), 1.87-1.96 (m, 2H), 1.48-1.58 (m, 2H), 1.24 (s, 24 H ), 0.85 (t, J = 3.4 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) δ: 150.36, 149.78, 147.65, 145.23, 142.86, 137.40, 133.02, 130.62, 126.68, 1, 123.21.4, 0.4, 3 , 108.36, 105.39, 102.05, 74.21, 57.01, 55.29, 31.23, 29.43, 28.99, 28.78, 28.64, 26.31, 25.22, 22.03, 13.87; ESI-MS m/z ⁺ : 546 [M-Br]

9-O-octadecylberberine ( B5 )

¹ H NMR (400 MHz, DMSO) δ: 9.75 (s, 1H), 8.94 (s, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H), 4.95 (t, J = 6.4 Hz, 2H), 4.28 (t, J = 6.4 Hz, 2H), 4.05 (s, 3H) , 3.23 (t, J = 5.3 Hz, 2H), 1.83-1.92 (m, 2H), 1.42-1.52 (m, 2H), 1.39-1.25 (m, 28H), 0.86 (t, J = 4.9 Hz, 3H ); ¹³ C NMR (101 MHz, DMSO) δ: 150.37, 149.80, 147.66, 145.22, 142.87, 137.44, 133.03, 130.63, 126.70, 123.26, 121.65, 120.40, 120.18, 108.37, 105.39, 102.04, 74.22, 57.01, 55.30 , 31.21, 29.41, 28.95, 28.75, 28.62, 26.31, 25.21, 22.01, 13.87; ESI-MS m/z : 574 [M-Br] ⁺ .

9-O-Benzylberberine ( B6 ) ¹ H NMR (400 MHz, DMSO) δ: 9.73 (s, 1H), 8.93 (s, 1H), 8.21 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.78 (s, 1H), 7.59 (d, J = 7.3 Hz, 2H), 7.38 (dt, J = 8.5, 5.1 Hz, 3H), 7.08 (s, 1H) , 6.17 (s, 2H), 5.36 (s, 2H), 4.91 (t, J = 6.7 Hz, 2H), 4.08 (s, 3H), 3.21 (t, J = 6.7 Hz, 2H); ¹³ C NMR ( 101 MHz, DMSO) δ: 150.66, 149.78, 147.63, 145.24, 141.95, 137.30, 136.40, 132.89, 130.57, 128.75, 128.37, 128.31, 126.49, 123.71, 121.77, 120.32, 120.15, 108.36, 105.39, 102.04, 75.32, 57.03 , 55.33, 26.31; ESI-MS m/z : 412 [M-Br] ⁺ .

9-O-decylberberine ( B12 ) ¹ H NMR (400 MHz, DMSO) δ 9.74 (s, 1H), 8.93 (s, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.78 (s, 1H), 7.08 (s, 1H), 6.16 (s, 2H), 4.94 (t, J = 6.3 Hz, 2H), 4.26 (t, J = 6.7 Hz, 2H), 4.04 (s, 3H), 3.20 (s, 3H), 1.91 – 1.81 (m, 2H), 1.45 (d, J = 7.3 Hz, 2H), 1.24 (s, 12H), 0.84 ( D, J = 6.8 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) Δ: 150.34, 149.72, 147.59, 145.23, 142.82, 137.32, 132.99, 130.57, 123.27, 120.38, 108.33, 105.399, 105.399, 105.39, , 102.03, 74.21, 56.99, 55.24, 31.26, 29.45, 28.96, 28.81, 28.67, 26.29, 25.23, 22.05, 13.90; ESI-MS m/z : 462 [M-Br] ⁺ .

2. Functional Validation of Berberine Derivatives

2.1 Cell Culture

C6 and U87 glioma cells were cultured in 10% fetal bovine serum (FBS) DMEM high-glucose medium under the conditions of 5% CO ₂ and 37°C. The culture medium was changed every 48 h and subcultured.

assay for cell viability

A control group and 5 drug groups with different concentrations were set up, with 3 replicate wells in each group. C6 cells in the logarithmic growth phase were digested and counted, seeded in a 96-well plate according to the number of cells 10,000/well, cultured in a 37°C, 5% _CO2 incubator for 24 h, and drug solutions of different concentrations were added. After continuing to culture for 24 h, 48 h, and 72 h, add 20 μL of tetrazolium salt solution (MTT, 5 mg/mL) to each well and continue to culture for 4 h, discard the supernatant, add 100 μL of DMSO to each well, and shake for 10 min, the absorbance (A) at 570 nm was measured on a microplate reader. Cell survival rate (%)= _{drug group} A/ _{control group} A×100%. The results are shown in Figure 1 and Figure 2. It can be seen from Figure 1 that the inhibitory effect of berberine and its derivatives on the proliferation of C6 cells was dose-dependent, and the difference in cell survival rate among different concentration groups (0, 5, 10, 20, 40 μM) was statistically significant ( P <0.05). Compared with the control group, berberine exhibited a significant inhibitory effect on proliferation when the concentration was greater than 20 μM ( P <0.05). The inhibitory effect of berberine derivatives (B3, B4, B5 and B6) on cell proliferation was less significant than that of berberine ( P < 0.05). Among them, B3 had the strongest inhibitory effect on proliferation, and 5 μM had a significant impact on the proliferation activity of cells, and the cell survival rate at each concentration was significantly lower than that of berberine and other derivatives ( P <0.05). Figure 2 shows the changes in cell viability after different concentrations of B3, B4, B5 and B6 acted on C6 cells for 24 h, 48 h and 72 h. The inhibitory effect of berberine derivatives on the proliferation of C6 cells was dose- and time-dependent ( P <0.05).

cell scratch test

Take C6 cells in the logarithmic growth phase and inoculate them on a 6-well plate, and place them in an incubator. After the cells cover about 90% of the monolayer, use a sterilized 20 mL pipette tip to evenly scratch the cells. Phosphate buffer saline (Phosphate Buffer Solution, PBS) was used to wash the cell debris three times, and replaced with serum-free culture medium. A control group and a drug group were set up, and the drug concentration in the culture medium of the drug group was 5 mmol/L. After 24 h, the degree of cell migration was observed with an inverted microscope (×40) and photographed. The cell migration distance was measured with Image J2x (version 2.1.4.7) software. According to the relative migration distance of cells = (0 h scratch width - 24 h scratch width) / 0 h scratch width × 100%, calculate the relative migration distance of each group, and then calculate the relative migration distance of the control group as 100%. Relative mobility of drug groups. The result is shown in Figure 3. Figure 3A shows that the cell scratches in the control group and berberine group were almost covered by the migrated cells after 24 h, covering more than 90% of the area; while the scratch area of each derivative group was still clearly visible. Comparing the relative mobility among the groups (Figure 3 B), there was a significant difference between the drug group and the control group, and the inhibitory effect of the derivative on migration was significantly stronger than that of the raw material berberine (P<0.05). Among them, B3 had the strongest inhibitory effect on the migration of C6 cells, and the relative migration rate (13.75%±4.86%) was significantly lower than that of other derivative groups (P<0.05). The relative migration rate of group B4 was 23.56%±8.67%, and its inhibitory effect on cell migration was second only to B3, but stronger than that of B5 and B6 (relative migration rates were 35.46%±2.33% and 46.89%±10.56%, respectively).

migration test

The cells were digested with trypsin and collected, washed three times in DMEM medium containing 10% FBS, and resuspended in serum-free DMEM medium (cell number 2.5×10 ⁵ /mL). Evenly add 100 μL of cell suspension to the upper chamber, and at the same time add appropriate concentration of drug solution; add 600 μL of DMEM culture solution containing 10% FBS to the lower chamber. At the same time, a 96-well plate with the same cell number was used for MTT experiment to detect the change of cell number. After _incubating at 37°C and 5% CO for 24 h, the upper chamber was removed, and the cells in the upper chamber were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 30 min, stained with 0.2% crystal violet staining solution for 10 min, washed three times with distilled water, and examined under a microscope ( ×200) randomly selected 9 fields of view to take pictures and count them; 3 small holes were set in parallel in each group, and the experiment was repeated 3 times. Finally, the migration ability of the cells was evaluated by the migration rate, and the migration rate = the number of migrated cells/the total number of MTT cells at the same concentration × 100%. The ratio of the mobility of each group to the mobility of the control group is the relative mobility. The result is shown in Figure 4. Figure 4A showed that the B3 and B4 groups had the least number of cells migrating to the lower chamber ( P <0.001).

Invasion test

Matrigel (BS Biosciences) 5 mL was thawed at 4 ℃ overnight, diluted with 4 ℃ pre-cooled serum-free DMEM culture medium according to DMEM:Matrigel=3:1, and 50 μL of the diluted Matrigel was added to the pre-cooled Transwell upper chamber, 37 Incubate at °C for 4 h to allow gel formation. The following operations are the same as those described in 2.4 (the amount of cells inoculated in the upper chamber is 5×10 ⁵ /100 μL). Finally, the invasion ability of the cells was evaluated by the invasion rate, where the invasion rate = the number of invasive cells/the total number of MTT cells at the same concentration × 100%. The ratio of the invasion rate of each group to the invasion rate of the control group was the relative invasion rate.

The result is shown in Figure 5. After drug treatment, the number of C6 cells passing through the recombinant artificial basement membrane was significantly less than that of the control group, and the invasion ability was inhibited, and the difference was statistically significant ( P <0.05). Among them, B3 and B4 (relative invasion rates were 40.78%±3.72% and 20.36%±5.84%, respectively) had significantly stronger inhibitory effect on invasion than the raw material berberine (relative migration rate was 56.17%±4.00%).

The above data were analyzed using SPSS 13.0 statistical software. Analysis of variance was used for the mean test, and P <0.05 was considered statistically significant.

Strong migration and invasion ability are the key factors restricting the long survival of patients with malignant glioma. However, a large number of current cytotoxic drugs not only cannot effectively inhibit the migration and invasion of glioma, but drug resistance and myelosuppressive effects limit their clinical application. The inventors found that the lipophilic derivatives of berberine provided by the present invention can increase the lipophilicity of berberine and at the same time enhance its inhibitory effect on the proliferation, migration and invasion of C6 glioma cells.

3. Mitochondrial targeting of berberine derivatives

C6 and U87 cells in the logarithmic growth phase were inoculated at 4×10 ⁵ cells/dish in glass-bottom culture dishes dedicated to laser confocal microscopy and cultured for 24 hours. The culture medium was removed, and the culture solution containing 1 μM berberine, 9-O-dodecyl berberine or 9-O-hexadecyl berberine was added respectively, and incubated for 24 hours. Aspirate the supernatant, wash three times with PBS (pH 7.4), stain with Mitotracker Green FM (500nM) for 30min, wash three times with ice-cold PBS, observe and take pictures under a laser confocal microscope.

It was observed under confocal laser that berberine and its lipophilic derivatives could selectively accumulate in mitochondria in C6 (Fig. 6 A) and U87 (Fig. 6 B) glioma cells. The mitochondria of C6 and U87 glioma cells were stained and localized by mitotracker green (green), and confocal laser observation was performed after incubation with berberine and its derivatives (yellow) for 24 hours. In all overlays (combined), there is good subcellular co-localization of yellow with green, which is shown as yellow-green when overlaid.

In C6 cells, berberine was more evenly distributed throughout the cell (including cytoplasm and nucleus), and the fluorescence intensity was weaker, showing weaker mitochondrial targeting. In comparison, the fluorescence distributions of berberine derivatives B3 and B4 are basically completely coincident with the green fluorescence of mitotracker, and there is basically no fluorescence in the nucleus, showing that berberine has stronger mitochondrial targeting. In U87 cells, B3 and B4 were not only accurately located in the mitochondria of the cells, but also had a strong fluorescence intensity, indicating that they were distributed to the mitochondria in large quantities, showing stronger mitochondrial targeting than in C6 cells.

Claims (9)

1. Use of a compound of formula (I) or a salt thereof in the manufacture of a medicament or a mitochondrial targeting delivery system for the treatment of glioblastoma:

a compound of the formula (I),

wherein R is C₁₀-C₁₈Alkyl, or benzyl.

2. Use according to claim 1, wherein R is C₁₀-C₁₈An n-alkyl group.

3. Use according to claim 1, wherein R is C₁₂-C₁₆An n-alkyl group.

4. Use according to claim 1, wherein R is n-dodecyl or n-hexadecyl.

5. Use according to any one of claims 1 to 4, wherein the salt is selected from the group consisting of hydrobromide, hydroiodide, hydrofluoride, hydrochloride, sulphate, nitrate, phosphate, citrate, acetate and lactate.

6. Use according to claim 1, wherein the compound is selected from: 9-O-n-decaalkyl berberine, 9-O-n-dodecyl berberine, 9-O-n-hexadecyl berberine, 9-O-n-octadecyl berberine, and 9-O-benzyl berberine.

7. The use of claim 1, wherein the medicament comprises at least one additional ingredient effective for the treatment of glioblastoma.

8. The use of claim 1, wherein the medicament comprises a pharmaceutically acceptable excipient.

9. The use of claim 1, wherein the mitochondrial targeting delivery system comprises at least one additional pharmaceutical ingredient.