COMPLEX SALT OF RUPESTONIC ACID AND ALKALOID, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

A complex salt formed from a compound of formula (I) and an alkaloid, a preparation method therefor and use thereof are provided. In particular, a complex salt formed from rupestonic acid and matrine, oxymatrine, sophocarpine and sophoridine, or a composition thereof, has potential efficacy in treating a tumor/cancer or preparing related medicaments.

##STR00001##

Claims

1. A complex salt formed from a compound of formula (I) and an alkaloid: ##STR00009## wherein the alkaloid is selected from matrine, oxymatrine, sophocarpine and sophoridine.

2. The complex salt according to claim 1, wherein the compound of formula (I) has a structure as shown in formula (II): ##STR00010##

3. The complex salt according to claim 1, wherein the complex salt is selected from a complex salt of rupestonic acid and matrine, a complex salt of rupestonic acid and oxymatrine, a complex salt of rupestonic acid and sophocarpine, and a complex salt of rupestonic acid and sophoridine, specifically having a structure shown as follows: ##STR00011##

4. The complex salt according to claim 1, wherein the compound of formula (I) and the alkaloid are in a molar ratio of 1:(0.5-5), such as 1:(0.8-2).

5. A preparation method for the complex salt according to claim 1, comprising the following steps: dissolving a compound of formula (I) and an alkaloid in an organic solvent, and mixing the solution to obtain the complex salt; wherein the compound of formula (I) and the alkaloid can be in a molar ratio of 1:(0.5-5), such as 1:(0.8-2).

6. The preparation method for the complex salt according to claim 5, wherein the organic solvent is selected from single solvents or mixed solvents that are capable of dissolving both of the compounds, such as methanol, ethanol, chloroform, acetone, toluene, dichloromethane and ethyl acetate.

7. A pharmaceutical composition comprising a therapeutically effective amount of the complex salt according to claim 1 or a composition thereof.

8. The pharmaceutical composition according to claim 8, further comprising one or more pharmaceutically acceptable auxiliary materials; wherein preferably, the pharmaceutical composition also further comprises one or more additional therapeutic agents.

9. A method for treating a tumor/cancer, comprising administering an effective amount of the complex salt according to claim 1 to a subject in need thereof.

10. The method according to claim 9, wherein the cancer is lung cancer, breast cancer, gastric cancer or cervical cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1: An HPLC chromatogram of matrine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0042] FIG. 2: An HPLC chromatogram of rupestonic acid, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0043] FIG. 3: An HPLC chromatogram of a complex salt of rupestonic acid and matrine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0044] FIG. 4: An HPLC chromatogram of oxymatrine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0045] FIG. 5: An HPLC chromatogram of a complex salt of rupestonic acid and oxymatrine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0046] FIG. 6: An HPLC chromatogram of sophocarpine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0047] FIG. 7: An HPLC chromatogram of a complex salt of rupestonic acid and sophocarpine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm;

[0048] FIG. 8: An HPLC chromatogram of sophoridine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm; and

[0049] FIG. 9: An HPLC chromatogram of a complex salt of rupestonic acid and sophoridine, with chromatographic conditions as follows: C.sub.18 column; mobile phase:V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm.

DETAILED DESCRIPTION

[0050] The technical scheme of the present disclosure will be further illustrated in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustration and explanation of the present disclosure, and should not be construed as limiting the protection scope of the present disclosure. All techniques implemented based on the above content of the present disclosure are encompassed within the protection scope of the present disclosure.

[0051] Unless otherwise stated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0052] Instruments and reagents:

[0053] Rupestonic acid (98%, provided by professor Haji Akber Aisa, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences); matrine, oxymatrine, sophocarpine and sophoridine (purity>98%, all purchased from Shanxi Yuning Biotechnology Co., Ltd.); LC3000N high performance liquid chromatograph (Beijing Chuangxintongheng Chromatography Technology Co., Ltd.); Hitachi L2000 high performance liquid chromatograph (Hitachi); CCK8 (Shanghai Beibo Biotechnology Co., Ltd.); DMEM high glucose medium (Thermo Fisher Scientific (Suzhou) Instruments Co., Ltd.); EDTA (pancreatin) (gibco); Foetal Bovine Serum (Biological Industries); phosphate buffered saline; 96-well cell culture plate; multifunctional microplate reader.

Example 1. Preparation of a Complex Salt of Rupestonic Acid and Matrine

[0054] ##STR00005##

[0055] 0.124 g (0.5 mmol) of rupestonic acid and 0.124 g (0.5 mmol) of matrine were added into a 20 mL round-bottom flask, followed by the addition of 5 mL of dried methanol, and the mixture was stirred at room temperature for 30 min. After the reaction was completed as detected by HPLC, the solution was concentrated under reduced pressure to obtain the complex salt of rupestonic acid and matrine (crude product). The crude product was purified by preparative HPLC to obtain a colorless sticky oil (chromatographic conditions: C.sub.18 column; mobile phase: V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm), HPLC purity: 98.9%.

Example 2. Preparation of a Complex Salt of Rupestonic Acid and Oxymatrine

[0056] ##STR00006##

[0057] 0.124 g (0.5 mmol) of rupestonic acid and 0.132 g (0.5 mmol) of oxymatrine were added into a 20 mL round-bottom flask, followed by the addition of 5 mL of dried methanol, and the mixture was stirred at room temperature for 30 min. After the reaction was completed as detected by HPLC, the solution was concentrated under reduced pressure to obtain the complex salt of rupestonic acid and matrine (crude product). The crude product was purified by preparative HPLC to obtain a colorless sticky oil (chromatographic conditions: C.sub.18 column; mobile phase: V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm), HPLC purity: 97.3%.

Example 3. Preparation of a Complex Salt of Rupestonic Acid and Sophocarpine

[0058] ##STR00007##

[0059] 0.124 g (0.5 mmol) of rupestonic acid and 0.123 g (0.5 mmol) of sophocarpine were added into a 20 mL round-bottom flask, followed by the addition of 5 mL of dried methanol, and the mixture was stirred at room temperature for 30 min. After the reaction was completed as detected by HPLC, the solution was concentrated under reduced pressure to obtain the complex salt of rupestonic acid and sophocarpine (crude product). The crude product was purified by preparative HPLC to obtain a colorless sticky oil (chromatographic conditions: C.sub.18 column; mobile phase: V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm), HPLC purity: 95.3%.

Example 4. Preparation of a Complex Salt of Rupestonic Acid and Sophoridine

[0060] ##STR00008##

[0061] 0.124 g (0.5 mmol) of rupestonic acid and 0.123 g (0.5 mmol) of sophoridine were added into a 20 mL round-bottom flask, followed by the addition of 5 mL of dried methanol, and the mixture was stirred at room temperature for 30 min. After the reaction was completed as detected by HPLC, the solution was concentrated under reduced pressure to obtain the complex salt of rupestonic acid and sophoridine (crude product). The crude product was purified by preparative HPLC to obtain a colorless sticky oil (chromatographic conditions: C.sub.18 column; mobile phase: V.sub.methanol:V.sub.water (containing 0.3% phosphoric acid), 7:3; detection wavelength: 254 nm), HPLC purity: 98.1%.

Example 5. In Vitro Anti-Tumor Activity Test

[0062] The obtained Artemisia Rupestris complex salts were subjected to an in vitro anti-tumor activity test, in which the in vitro inhibitory activity of the Artemisia Rupestris complex salts on the lung adenocarcinoma cell line (A549) and cervical carcinoma cell line (Hela) was mainly researched. The specific test process was illustrated by the process for testing the lung adenocarcinoma cell line (A549):

[0063] 1. Preparation of Test Sample

[0064] The prepared Artemisia Rupestris complex salt was weighed, added into a 5 mL plastic centrifuge tube and diluted to

[0065] 1 mL with DMSO, to obtain a test sample at an initial concentration. Then the test sample at an initial concentration was serially diluted with DMSO to obtain test samples in 5 different concentration gradients in sequence, which were stored in a refrigerator at 4° C. for later use.

[0066] 2. Incubation and inhibitory activity test of the lung adenocarcinoma cell line (A549)

[0067] The lung adenocarcinoma cell line (A549) was incubated in an incubator for 24 h under the conditions of 37° C., 5% CO.sub.2, and saturated humidity. When the cells were in logarithmic growth phase, the culture supernatant was discarded, and the residue was digested with 0.25% trypsin-EDTA solution. Then, the digestion was terminated with high glucose medium. The cells were seeded in a 96-well plate with a cell density of 5000 cells/well. The 96-well plate was incubated in an incubator for 24 h. Then the cell culture supernatant in the 96-well plate was discarded. 100 μL of high glucose medium was added to the 96-well plate, and test samples at different concentrations were added to wells at 1 μL/well (5 duplicate wells were set for each concentration). After the plate was incubated in an incubator for 48 h under the conditions of 37° C., saturated humidity and 5% CO.sub.2, 10 μL of CCK8 was added to each well, and the plate was then incubated in the incubator at 37° C. for 1-4 h. The absorbance of each well at 450 nm was measured on a multifunctional microplate reader. Inhibition rate %=[(OD.sub.control cells−OD.sub.treatment cells)/(OD.sub.control cells−OD.sub.blank)]×100. Negative control was a mixed solution of V.sub.high glucose medium and V.sub.DMSO at a ratio of 10:1.

[0068] The process for testing the inhibition of those compounds on the cervical cancer cell line (Hela) was the same as above. The results of the inhibition on the two tumor cell lines are shown in Table 1.

TABLE-US-00001 TABLE 1 Test results of the activity of the compounds on two tumor cell lines IC.sub.50 (μM) Compound A549 Hela Rupestonic acid Inactive Inactive Matrine 6.37 × 10.sup.3 774.0 Oxymatrine 7.32 × 10.sup.5 0.95 Sophocarpine 240.9 121.7 Sophoridine  1.6 × 10.sup.4 1.49 × 10.sup.3 Complex salt of rupestonic acid and   1.492 11.78 matrine Complex salt of rupestonic acid and / 25.17 oxymatrine Complex salt of rupestonic acid and 713.7 114.8 sophocarpine Complex salt of rupestonic acid and  61.26 17.90 sophoridine

[0069] Examples of the present disclosure have been described above. However, the present disclosure is not limited to the above examples. Any modification, equivalent, improvement and the like made without departing from the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.