CN104211697B - Berberine derivatives and uses thereof - Google Patents

Abstract

The compound or its salt of formula (I) of the present invention and their purposes, wherein R is C₁₀‑C₁₈Alkyl or benzyl,

Description

Berberine derivatives and uses thereof

technical field

The present invention relates to berberine derivatives and their preparation methods and uses.

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 a compound of formula (I) or a salt thereof

Formula (I),

Wherein, 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 preferably, R is C ₁₀ -C ₁₄ n-alkyl. More preferably, R is n-decyl or n-dodecyl.

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: 13-n-decyl berberine, 13-n-dodecyl berberine, 13-n-hexadecyl berberine, 13-n-decyl berberine Octyl berberine, and 13-benzyl berberine.

Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a salt thereof and a pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition may further comprise at least one additional ingredient effective for treating malignant glioma for use in combination with the derivative of the present invention.

According to the third aspect of the present invention, the present invention provides the use of the compound of the above formula (I) or a salt thereof or a pharmaceutical composition in the preparation of a drug for treating malignant glioma.

According to the fourth 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.

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

The inventors found that 13-substituted long-chain alkyl or benzyl berberine derivatives are unexpectedly better than other berberine derivatives and berberine derivatives for malignant glioma cell growth inhibition, migration inhibition and invasion inhibition. the base 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, it improves the lipid solubility of the compound and effectively transports the compound across the biomembrane to the lesion by improving transmembrane transport sites and cells, thereby enhancing the efficacy.

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 ₁₀ -C ₁₄ alkyl refers to having 10 to 14 carbon atoms Straight chain or branched chain alkyl.

Description of drawings

Figure 1 shows the migration ability of C6 glioma cells detected by Transwell migration assay (`x±s, n=3; * means compared with the control group, P<0.05); B7 represents 13-decyl berberine; B8 Represents 13-dodecyl berberine.

Figure 2 shows the invasion ability of C6 glioma cells detected by Transwell invasion assay (`x±s, n=3; * indicates that compared with the control group, P<0.05); B7 represents 13-decyl berberine; B8 Represents 13-dodecyl berberine.

Figure 3 shows the migration ability of U87 glioma cells detected by Transwell migration assay (`x±s, n=3; * indicates that compared with the control group, P<0.05); B12 represents 9-O-decyl berberine ; B7 represents 13-decyl berberine.

Figure 4 shows the invasion ability of U87 glioma cells detected by Transwell invasion assay (`x±s, n=3; * means P<0.05 compared with the control group); B12 means 9-O-decyl berberine ; B7 represents 13-decyl berberine.

Fig. 5 shows confocal observation results of mitochondrial localization of berberine lipophilic derivatives in C6 (A) and U87 (B) glioma cells. mitotracker means mitochondria marked with mitotracker green, showing green fluorescence; drug means berberine and its lipophilic derivatives, showing yellow fluorescence; overlay means the superposition of the above two images. B8 represents 13-dodecyl berberine; B9 represents 13-hexadecyl berberine.

detailed description

1. Synthesis and identification of 13-substituted berberine derivatives

1.1 Synthesis and identification of intermediate dihydroberberine (Dihydroberberine, 1b)

Intermediate 1b was prepared by reduction method.

Dry 16 g (0.0162 mol) was suspended in 50 mL pyridine and stirred at room temperature. Slowly add 700 mg of NaBH ₄ , when the reddish-brown clear solution becomes clear, immediately add 200 mL of ice water to the reaction solution, filter to obtain intermediate 1b, which appears as a light yellow powder, and vacuum-dry at 30 °C for use. ESI-MS m/z: 338 [M+H] ⁺ .

1.2 Synthesis and identification of 13-decyl berberine (B7)

Bromidedecane 0.89 mL (4 eq is 4 times the molar amount of 1b), sodium iodide (NaI) 0.60 g (4 eq), anhydrous acetonitrile 20 mL, put in a sealed high-pressure reaction bottle, and react at 80 °C 24 h. Add 0.34 g (1 mmol, 1 eq) of 1b, fill with N ₂ for 15 min, and react for 12 h. After that, it was heated to reflux at 60 °C for 2 h to fully oxidize it. Cool to room temperature and filter. The filtrate was spin-dried and passed through the column three times in sequence: neutral alumina column (petroleum ether-n-butanol 10:1), silica gel column (supernatant of n-butanol-water-glacial acetic acid 50:3:3), gel Gel column (methanol-dichloromethane-anhydrous ether 1:1:1). Yellow crystal 7 was obtained with a yield of about 15%. ¹ H NMR (400 MHz, DMSO) δ: 9.90 (s, 1H), 8.21 (s, 2H), 7.31 (s, 1H), 7.17 (s,1H), 6.20 (s, 2H), 4.81 (t, J=5.6Hz, 2H), 4.11 (s, 3H), 4.10 (s, 3H), 3.34(m, 2H), 3.09 (t, J=5.6Hz, 2H), 1.74 (m, 2H), 1.38 ( m, 2H), 1.24 (m, 12H), 0.87 (t, J = 6.6 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) δ: 150.69, 149.49, 147.03, 144.90, 144.78, 136.24, 134.65, 134.56 , 132.68, 126.41, 121.91, 121.71,120.78, 109.58, 108.85, 102.60, 62.54, 57.49, 31.76, 30.88, 29.12, 27.87, 22.55, ESI-MS mi-476 [mi] ⁺ .

1.3 Synthesis and identification of 13-dodecylberberine (B8)

Bromododecane 1.00 mL (4 eq), NaI 0.60 g (4 eq), and anhydrous acetonitrile 20 mL were placed in a sealed high-pressure reaction bottle, and reacted at 80 °C for 24 h. Add 0.34 g (1 mmol, 1 eq) of 1b, fill with N ₂ for 15 min, and react for 12 h. After that, it was heated to reflux at 60 °C for 2 h to fully oxidize it. Cool to room temperature and filter. The filtrate was spin-dried and passed through the column three times in sequence: neutral alumina column (petroleum ether-n-butanol 10:1), silica gel column (supernatant of n-butanol-water-glacial acetic acid 50:3:3), gel Gel column (methanol-dichloromethane-anhydrous ether 1:1:1). Yellow crystal 8 was obtained with a yield of about 10%. ¹ H NMR (400 MHz, DMSO)δ: 9.92 (s, 1H), 8.20 (s, 2H), 7.29 (s, 1H), 7.16 (s, 1H), 6.18 (s, 2H), 4.81(t, J=5.6Hz, 2H), 4.10 (s, 3H), 4.09 (s, 3H), 3.34 (m, 2H), 3.08 (t, J=5.6Hz,2H), 1.75 (m, 2H), 1.37 ( m, 2H), 1.23 (m, 16H), 0.85 (m, 3H); ¹³ C NMR (101MHz, DMSO) δ: 150.69, 149.50, 147.04, 144.81, 144.75, 136.51, 136.26, 134.669, 132.57.50, 93 , 121.91, 121.71, 120.78, 109.60, 108.83, 102.59,62.51, 57.47, 31.76, 30.86, 29.47, 29.40, 29.16, 29.06, ^27.87,22.56 .

1.4 Synthesis and identification of 13-hexadecyl berberine (B9)

Bromohexadecane 1.23 mL (4 eq), NaI 0.60 g (4 eq), and anhydrous acetonitrile 20 mL were placed in a sealed high-pressure reaction bottle, and reacted at 80 °C for 24 h. Add 0.34 g (1 mmol, 1 eq) of 1b, fill with N ₂ for 15 min, and react for 12 h. After that, it was heated to reflux at 60 °C for 2 h to fully oxidize it. Cool to room temperature and filter. The filtrate was spin-dried and passed through the column three times in sequence: neutral alumina column (petroleum ether-n-butanol 500:75), silica gel column (supernatant of n-butanol-water-glacial acetic acid 50:3:3), condensation Gel column (methanol-dichloromethane-anhydrous ether 1:1:1). Yellow crystal 9 was obtained with a yield of about 10%. ¹ H NMR (400 MHz, DMSO)δ: 9.90 (s, 1H), 8.20 (s, 2H), 7.29 (s, 1H), 7.16 (s, 1H), 6.18 (s, 2H), 4.80(t, J=5.6Hz, 2H), 4.10 (s, 3H), 4.09 (s, 3H), 3.34 (s, 2H), 3.08 (m, 2H),1.77 – 1.71 (m, 2H), 1.36 (m, 2H ), 1.23 (s, 24H), 0.86 (t, J = 5.8 Hz, 3H). ¹³ C NMR (101 MHz, DMSO) δ: 150.16, 148.98, 146.51, 144.29, 135.70, 134.09,134.01, 532.17, 131. 131.40, 128.57, 125.88, 121.31, 121.19, 120.22,108.99, 108.31, 102.06, 61.98, 56.94, 31.24, 30.38, 29.96, 29.00, 28.96,28.65, 28.61, 28.40, 22.03, 18.59, 13.83, 13.45.ESI-MS m /z: 560 [MI] ⁺ .

1.5 Synthesis and identification of 13-octadecylberberine (B10)

Bromooctadecane 1.35 mL (4 eq), NaI 0.60 g (4 eq), and anhydrous acetonitrile 20 mL were placed in a sealed high-pressure reaction bottle, and reacted at 80 °C for 24 h. Add 0.34g (1 mmol, 1 eq) of 1b, fill with N ₂ for 15 min, and react for 12 h. After that, it was heated to reflux at 60 °C for 2 h to fully oxidize it. Cool to room temperature and filter. The filtrate was spin-dried and passed through the column three times in sequence: neutral alumina column (petroleum ether-n-butanol 500:65), silica gel column (supernatant of n-butanol-water-glacial acetic acid 50:3:3), condensation Gel column (methanol-dichloromethane-anhydrous ether 1:1:1). Yellow crystal 10 was obtained with a yield of about 10%. ¹ H NMR (400 MHz,DMSO) δ: 9.90 (s, 1H), 8.20 (s, 2H), 7.29 (s, 1H), 7.16 (s, 1H), 6.18 (s,2H), 4.80 (s, 2H), 4.10 (s, 3H), 4.09 (s, 3H), 3.24 (d, J = 7.8 Hz, 2H), 3.08(s, 2H), 1.83 – 1.70 (m, 2H), 1.37 (m, 2H ), 1.23 (s, 28H), 0.85 (t, J = 6.8Hz, 3H). ¹³ C NMR (101 MHz, DMSO) δ: 150.67, 149.49, 147.03, 144.81, 144.74,136.25, 134.67, 134.58, 132. 126.38, 121.89, 121.70, 120.76, 109.58, 108.82, ^102.58 , 62.50, 57.47, 31.75, 30.87, 29.48, 29.16, 27.87, 22.55, 14.41.

1.6 Synthesis and identification of 13-benzylberberine (B11)

0.48 mL (4 eq) of benzyl bromide, 0.60 g (4 eq) of NaI, and 20 mL of anhydrous acetonitrile were placed in a sealed high-pressure reaction bottle and reacted for 24 h. Add 0.34 g (1 mmol, 1 eq) of 1b, fill with N ₂ for 15 min, and react for 12 h. After that, it was heated to reflux at 60 °C for 2 h to fully oxidize it. Cool to room temperature and filter. The filtrate was spin-dried and passed through the column three times in sequence: neutral alumina column (petroleum ether-n-butanol 500:70), silica gel column (supernatant of n-butanol-water-glacial acetic acid 50:3:3), condensate Gel column (methanol-dichloromethane-anhydrous ether 1:1:1). Yellow crystal 11 was obtained with a yield of about 37%. ¹ H NMR (400 MHz, DMSO) δ: 10.06 (s, 1H), 8.11 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 9.3 Hz, 1H), 7.38 (t, J = 7.3 Hz , 2H), 7.31 (d, J = 7.1 Hz, 1H), 7.18 (s, 3H), 6.98 (s, 1H), 6.09 (s,2H), 4.90 (s, 2H), 4.76 (s, 2H) , 4.13 (s, 3H), 4.04 (s, 3H), 3.17 (s, 2H).13C NMR (101 MHz, DMSO) δ: 150.69, 149.69, 146.88, 145.99, 144.74, 139.63,137.64, 134.523, 13 130.48, 129.57, 128.49, 127.27, 126.64, 122.15, 121.74, 120.49, 108.97, ^108.60 , 102.53, 62.55, 57.41, 35.98, 27.76.

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.

migration test

Trypsinize and collect C6 or U87 cells, wash 3 times in DMEM medium containing 10% FBS, and resuspend 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, where the migration rate = the number of migrated cells/the total number of cells in MTT at the same concentration × 100%. The ratio of the mobility of each group to the mobility of the control group is the relative mobility.

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 2.2 (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 cells in MTT 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 results are shown in Figure 1. The number of cells migrating to the lower chamber in the compound B7 and compound B8 groups was less than that in the control group (P<0.05), and the number of migrating cells in the compound B7 was the least.

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, the inhibitory effect of compound B7 and compound B8 on invasion was significantly stronger than that of the control group (raw material berberine), and the inhibitory effect of compound B7 on invasion was more obvious (Figure 2).

As shown in Figure 3, the number of cells migrating to the lower chamber in the compound B12 (9-O-n-decane berberine) and compound B7 groups was less than that in the control group (P<0.05), and the number of migrating cells in the compound B7 was the least .

As shown in Figure 4. After drug treatment, the number of U87 glioma 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, the inhibitory effect of compound B12 (9-O-n-decane berberine) and compound B7 on invasion was significantly stronger than that of the control group (raw material drug berberine), and the inhibitory effect of compound B7 on invasion was more obvious.

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 in the lipophilic berberine derivatives provided by the present invention, increasing the lipophilicity of berberine can enhance its inhibitory effect on the proliferation, migration and invasion of C6 and U87 glioma cells. The mechanism is not clear, and the possible reason is that, compared with berberine, the transmembrane effect of the derivative on the biomembrane is enhanced, thereby improving the physiological activity.

3. Mitochondrial targeting of berberine derivatives

C6 or U87 cells in the logarithmic growth phase were inoculated at 4×10 ⁵ cells/dish in a special glass-bottom culture dish for laser confocal microscopy and cultured for 24 hours. The culture medium was removed, and the culture solution containing 1 μM berberine, 13-dodecyl berberine or 13-dodecyl berberine was added respectively, and incubated for 24 hours. Aspirate the supernatant, wash three times with PBS (pH 7.4), stain with Mitotracker GreenFM (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 co-localize with mitochondria in C6 (Fig. 5A) and U87 (Fig. 5B) 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 the cytoplasm and nucleus), and the fluorescence intensity was weaker, showing weaker mitochondrial targeting. In comparison, the fluorescence distributions of berberine derivatives B8 (13-dodecyl berberine) and B9 (13-dodecyl berberine) are basically completely coincident with the green fluorescence of mitotracker, and there is basically no fluorescence in the nucleus Fluorescence, showing greater mitochondrial targeting of lesser berberine. In U87 cells, B8 and B9 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 (3)

1. the compound or its salt of formula (I) purposes in preparation treatment glioblastoma tumor medicine,

Formula (I),

Wherein R is decane base or dodecyl.

Purposes the most according to claim 1, wherein said salt is selected from hydrobromate, hydriodate, hydrofluoride, hydrochloric acid Salt, sulfate, nitrate, phosphate, citrate, acetate and lactate.

Purposes the most according to claim 1, wherein said glioblastoma tumor medicine also comprises pharmaceutical excipient.