Ferrocene formic acid and alkaloid double salts, preparation methods and uses thereof

Abstract

The present invention provides a ferroceneformic acid matrine, or a ferroceneformic acid oxymatrine, or a ferroceneformic acid sophoridine double salt. Through testing, it is found that the obtained double salt has anti-tumor efficacy. Moreover, the preparation method of the obtained double salt is simple and easy to purify, so it has certain application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a double salt formed by ferrocene formic acid and alkaloids, a preparation method thereof, and its use in the treatment of tumors / cancers. Background Art

[0002] Cancer has become the most important lethal disease worldwide. Cancer can occur in various organs and tissues at any age. The main types of cancer leading to death include: lung cancer, colorectal cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, thyroid cancer, pancreatic cancer, prostate cancer, cervical cancer, etc. Tumors with extremely high incidence and fatality rates have seriously affected human health and survival. Although some small molecule anti-cancer drugs have been used clinically and some compounds are undergoing pre-clinical studies. However, most cancer patients are already in the middle to late stage when they discover their condition, and the overall clinical treatment effect is poor. Especially the continuous emergence of multi-drug resistance makes the treatment of cancer extremely difficult. Therefore, it is urgent to develop new anti-cancer drugs with high activity and low side effects to meet the clinical needs. Traditional Chinese medicine has a long history of use in China and relatively low toxicity and side effects. Structural modification of active monomer compounds found in traditional Chinese medicine to improve their pharmacological activities has become the mainstream of innovative drug research and development today. However, how to develop new anti-cancer drugs with high activity and low side effects is a major difficulty at the present stage. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides the following ferrocene formic acid matrine, or ferrocene formic acid oxymatrine, or ferrocene formic acid sophoridine double salt:

[0004]

[0005] The present invention also provides a preparation method of the above double salt, including: mixing ferrocene formic acid and matrine, or ferrocene formic acid and oxymatrine, or ferrocene formic acid and sophoridine in a molar ratio of 1:1, and carrying out a stirring reaction or a reflux reaction or an ultrasonic reaction at 0 - 30 °C in an organic solvent.

[0006] The organic solvent is: a single solvent or a mixed solvent such as methanol, ethanol, chloroform, acetone, toluene, dichloromethane, ethyl acetate, acetonitrile, etc. that can dissolve the two compounds.

[0007] According to the embodiment of the present invention, the organic solvent is more preferably methanol or acetonitrile.

[0008] According to the embodiment of the present invention, the reaction is carried out under the protection of an inert gas, for example, under nitrogen protection.

[0009] The present invention also provides the use of one or a combination of the above-mentioned ferrocenecarboxylic acid matrine complex salts, oxidized matrine complex salts, and sophoridine complex salts in the preparation of a medicament for treating tumors.

[0010] According to an embodiment of the present invention, the tumor is cancer.

[0011] According to an embodiment of the present invention, the cancer is gastric cancer, breast cancer, lung adenocarcinoma, and cervical cancer.

[0012] The present invention also provides a pharmaceutical composition, which comprises one or a combination of the above-mentioned ferrocenecarboxylic acid matrine complex salts, ferrocenecarboxylic acid oxidized matrine complex salts, and ferrocenecarboxylic acid sophoridine complex salts.

[0013] According to an embodiment of the present invention, the pharmaceutical composition is used for treating tumors.

[0014] According to an embodiment of the present invention, the tumor is cancer.

[0015] According to an embodiment of the present invention, the pharmaceutical composition can be prepared into any pharmaceutically acceptable formulation form, such as solid oral preparations, injection preparations, emulsions, creams, tinctures, and ointments.

[0016] According to an embodiment of the present invention, the pharmaceutical composition can be a solid oral preparation or an injection preparation.

[0017] According to an embodiment of the present invention, the solid oral preparation can be capsules, tablets, granules, sustained-release agents, etc.

[0018] According to an embodiment of the present invention, the injection can be a small needle injection or a freeze-dried injection.

[0019] According to an embodiment of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; the excipients include one or a mixture of several of fillers, binders, disintegrants, lubricants, flavoring agents, enteric coatings, or sustained-release materials.

[0020] According to an embodiment of the present invention, the fillers include one or a mixture of several of lactose, sucrose, dextrin, starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium carbonate, and microcrystalline cellulose;

[0021] According to an embodiment of the present invention, the binders include one or a mixture of several of sucrose, starch, sodium carboxymethylcellulose, methylcellulose, polyethylene glycol, and water;

[0022] According to an embodiment of the present invention, the disintegrants include one or a mixture of several of starch, crospovidone, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and sodium carboxymethylcellulose;

[0023] According to an embodiment of the present invention, the lubricant includes a mixture of one or more of talc powder, magnesium stearate, stearic acid, colloidal silica, polyethylene glycol - 4000, and polyethylene glycol - 6000;

[0024] According to an embodiment of the present invention, the flavoring agent includes a mixture of one or more of sucrose, sorbitol, sodium saccharin, maltitol, stevioside, and aspartame;

[0025] According to an embodiment of the present invention, the sustained - release material includes a mixture of one or more of hydroxypropyl methylcellulose and guar gum with different viscosity specifications.

[0026] According to an embodiment of the present invention, when the pharmaceutical composition is a solid oral preparation, it includes the following components in parts by weight: 1 - 5 parts of the ferrocene formic acid alkaloid double salt, 2 - 20 parts of lactose, 1 - 10 parts of pre - gelatinized starch, 1 - 5 parts of microcrystalline cellulose, 0.1 - 5 parts of sodium carboxymethyl starch, 2 - 10 parts of 10% povidone solution, 1 - 5 parts of magnesium stearate, and 1 - 5 parts of talc powder.

[0027] According to an embodiment of the present invention, when the pharmaceutical composition is a solid oral preparation, it includes the following components in parts by weight: 2 parts of the ferrocene formic acid alkaloid double salt, 13 parts of lactose, 3.5 parts of pre - gelatinized starch, 2.5 parts of microcrystalline cellulose, 0.8 parts of sodium carboxymethyl starch, 5 parts of 10% povidone solution, 1.5 parts of magnesium stearate, and 1.5 parts of talc powder.

[0028] According to an embodiment of the present invention, when the pharmaceutical composition is a solid oral preparation, its preparation method includes: mixing the excipients and passing through a 40 - 100 - mesh sieve; adding the ferrocene formic acid alkaloid double salt to the 10% povidone solution, mixing, then adding to the sieved excipients, grinding and mixing, drying at 50 - 60 °C for 2 - 3 hours, and tabletting to obtain the solid oral preparation of the pharmaceutical composition.

[0029] Beneficial effects

[0030] The present invention provides a ferrocene formic acid alkaloid double salt represented by formula (I), its preparation method and uses. By testing the double salts of ferrocene formic acid with matrine, oxymatrine, and sophoridine respectively, it is found that they have anti - tumor effects. The double salt or its composition has the potential efficacy for treating diseases such as tumors / cancers.

[0031] Moreover, the obtained double salt has a simple preparation method, is easy to purify, and has certain application prospects. Description of the drawings

[0032] Figure 1 : HPLC chromatogram of matrine, chromatographic conditions: C 18 Column; mobile phase: V甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0033] Figure 2 : HPLC chromatogram of ferroceneformic acid. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0034] Figure 3 : HPLC chromatogram of the complex salt of ferroceneformic acid and matrine. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0035] Figure 4 : HPLC chromatogram of oxymatrine. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0036] Figure 5 : HPLC chromatogram of the complex salt of ferroceneformic acid and oxymatrine. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0037] Figure 6 : HPLC chromatogram of sophoridine. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm;

[0038] Figure 7 : HPLC chromatogram of the complex salt of ferroceneformic acid and sophoridine. Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 : V 水(含0.3％磷酸和2％THF) , 7:3; Detection wavelength: 254 nm. Detailed implementation manners

[0039] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0041] Instruments and Reagents:

[0042] Ferrocene carboxylic acid (99%, purchased from Bidepharm); matrine, oxymatrine, and sophocarpine were all purchased from Shanxi Yuning Biotechnology Co., Ltd. (purity > 98%); LC3000N high-performance liquid chromatograph (Beijing Innovation Tongheng Chromatography Technology Co., Ltd.); Hitachi L2000 high-performance liquid chromatograph (Hitachi HITACHI); CCK8 (Shanghai Beyotime Biotechnology Co., Ltd.); high-glucose DMEM medium (Thermo Fisher Scientific (Suzhou) Instruments Co., Ltd.); EDTA (trypsin) (gibco); Foetal Bovine Serum (Biological Industries); phosphate buffer solution; 96-well cell culture plate; multifunctional microplate reader.

[0043] Example 1: Preparation of Ferrocene Carboxylic Acid - Matrine Double Salt

[0044]

[0045] 0.115 g (0.5 mmol) of ferrocene carboxylic acid and 0.124 g (0.5 mmol) of matrine were added to a 20 mL round-bottom flask, 5 mL of dry chromatographic methanol was added, and the reaction was refluxed under nitrogen protection. After the reaction was completed as detected by HPLC, the crude product of ferrocene carboxylic acid - matrine double salt was obtained by concentration under reduced pressure. This crude product was separated and purified by preparative HPLC to obtain a dark red solid (chromatographic conditions: C 18 column; mobile phase: V 甲醇 :V 水(含0.3％磷酸) , 7:3; detection wavelength: 254 nm), retention time: 10.883 min, HPLC purity: 98.5%.

[0046] Example 2: Preparation of Ferrocene Carboxylic Acid - Oxymatrine Double Salt

[0047]

[0048] 0.115 g (0.5 mmol) of ferrocene carboxylic acid and 0.132 g (0.5 mmol) of oxymatrine were added to a 20 mL round-bottom flask, 5 mL of dry chromatographic methanol was added, and the reaction was refluxed under nitrogen protection. After the reaction was completed as detected by HPLC, the crude product of ferrocene carboxylic acid - oxymatrine double salt was obtained by concentration under reduced pressure. This crude product was separated and purified by preparative HPLC to obtain a dark red solid (chromatographic conditions: C 18 column; mobile phase: V 甲醇 :V 水(含0.3％磷酸), 7:3; Detection wavelength: 254 nm), Retention time: 10.840 min, HPLC purity: 98.4%.

[0049] Example 3: Preparation of the ferroceneformic acid sophoridine complex salt

[0050]

[0051] Add 0.115 g (0.5 mmol) of ferroceneformic acid and 0.124 g (0.5 mmol) of sophoridine to a 20 mL round-bottom flask, add 5 mL of dry chromatographic methanol, and reflux the reaction under nitrogen protection. After the reaction is completed as detected by HPLC, concentrate under reduced pressure to obtain the crude product of the ferroceneformic acid sophoridine complex salt. The crude product is separated and purified by preparative HPLC to obtain a pale yellow solid (Chromatographic conditions: C 18 column; Mobile phase: V 甲醇 :V 水(含0.3％磷酸) , 7:3; Detection wavelength: 254 nm), Retention time: 10.860 min, HPLC purity: 98.8%.

[0052] The appearance states and physical property constants of all raw materials and target compounds in Examples 1-3 are shown in Table 1.

[0053] Table 1 Appearance states and physical property constants of raw materials and target compounds in Examples 1-3

[0054]

[0055]

[0056] Due to the properties of the ferroceneformic acid sophoridine complex salt in the above table, the melting point test could not be carried out, so "-" indicates not tested.

[0057] Example 4: In vitro anti-tumor activity test

[0058] The prepared ferroceneformic acid alkaloid complex salt was subjected to an in vitro anti-tumor activity test, and its in vitro inhibitory activities against gastric cancer cell lines, breast cancer cell lines (MCF-7), lung adenocarcinoma cell lines (A549), and cervical cancer cell lines (Hela) were mainly studied. The specific test process is described by taking the test process of breast cancer MCF-7 cell lines as an example:

[0059] 1. Preparation of test sample concentrations

[0060] (A) Weigh 24.3 mg of the matrine complex salt of ferrocenecarboxylic acid, dilute it with 1 mL of DMSO to obtain an initial concentration of 24.3 mg / mL, and then perform serial dilution of the initial concentration with DMSO to successively obtain 6 different concentration gradients of 12.15 mg / mL, 6.075 mg / mL, 3.0375 mg / mL, 1.5188 mg / mL, 0.7594 mg / mL, and 0.3797 mg / mL, and store them in a 4°C refrigerator for later use.

[0061] (B) Weigh 25.1 mg of the oxymatrine complex salt of ferrocenecarboxylic acid, dilute it with 1 mL of DMSO to obtain an initial concentration of 25.1 mg / mL, and then perform serial dilution of the initial concentration with DMSO to successively obtain 6 different concentration gradients of 12.55 mg / mL, 6.275 mg / mL, 3.1375 mg / mL, 1.5688 mg / mL, 0.7844 mg / mL, and 0.3922 mg / mL, and store them in a 4°C refrigerator for later use.

[0062] (C) Weigh 29.6 mg of the sophoridine complex salt of ferrocenecarboxylic acid, dilute it with 1 mL of DMSO to obtain an initial concentration of 29.6 mg / mL, and then perform serial dilution of the initial concentration with DMSO to successively obtain 6 different concentration gradients of 14.8 mg / mL, 7.4 mg / mL, 3.7 mg / mL, 1.85 mg / mL, 0.925 mg / mL, and 0.4625 mg / mL, and store them in a 4°C refrigerator for later use.

[0063] 2. Culture and inhibition activity test of breast cancer cell line (MCF-7)

[0064] Place the breast cancer cell line MCF-7 in an incubator at 37°C, saturated humidity, and containing 5% CO 2 for 24 hours. When the cells are in the logarithmic growth phase, aspirate the upper culture medium, digest with 0.25% trypsin-EDTA solution, and then terminate the digestion with high-glucose medium. Inoculate the cells into a 96-well plate so that the cell density is 5000 cells / well. Place the 96-well plate in the incubator and culture for 24 hours. Then aspirate the cell culture medium in the 96-well plate. Add 100 μL of high-glucose medium to the 96-well plate, and then add 1 μL of the test sample with different concentrations to each well (set 5 replicates for each concentration). Then place it in an incubator at 37°C, saturated humidity, and 5% CO 2 and continue to culture for 48 h. Then add 10 μL of CCK8 to each well and continue to incubate in a 37°C incubator for 1 - 4 h. Measure the absorbance value of each well at a wavelength of 450 nm on a multifunctional microplate reader. According to the inhibition rate % = [(OD 对照细胞 - OD 加药细胞 ) / (OD 对照细胞-OD 空白 )]×100. The negative control is V 高糖培养基 / V DMSO : a mixed solution of 10:1.

[0065] The testing process of the ferrocene formic acid alkaloid double salts against gastric cancer cell lines, lung adenocarcinoma cell lines (A549), and cervical cancer cell lines (Hela) is the same as that against breast cancer cell lines (MCF-7).

[0066] The ferrocene alkaloid double salts have strong inhibitory activity against breast cancer cell lines (MCF-7), and the inhibition results are shown in Table 2.

[0067] Table 2 Inhibition results of ferrocene formic acid alkaloid double salts against breast cancer cell lines (MCF-7)

[0068]

[0069]

[0070] "-" did not show inhibitory activity in this experiment.

[0071] From the above results, it can be seen that the ferrocene formic acid alkaloid double salts of the present invention have strong inhibitory activity against tumor cell lines and have significant advantages over the activities of the alkaloid raw materials.

[0072] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Matrine ferrocenecarboxylate double salt, or oxidized matrine ferrocenecarboxylate double salt, or sophoridine ferrocenecarboxylate double salt:

2. Preparation method of the double salt according to claim 1, characterized in that, comprising: Mixing ferrocenecarboxylic acid and matrine, or ferrocenecarboxylic acid and oxidized matrine, or ferrocenecarboxylic acid and sophoridine in a molar ratio of 1:1, and stirring, refluxing or sonicating in an organic solvent at 0 - 30 °C.

3. The preparation method according to claim 2, characterized in that, the organic solvent is: a single solvent or a mixed solvent of methanol, ethanol, chloroform, acetone, toluene, dichloromethane, ethyl acetate, acetonitrile.

4. The preparation method according to claim 2, characterized in that, the reaction is carried out under the protection of an inert gas.

5. Use of one or a combination of the matrine ferrocenecarboxylate double salt, or oxidized matrine ferrocenecarboxylate double salt, or sophoridine ferrocenecarboxylate double salt according to claim 1 in the preparation of a drug for treating breast cancer.

6. A pharmaceutical composition, characterized in that, it comprises one or a combination of the matrine ferrocenecarboxylate double salt, oxidized matrine ferrocenecarboxylate double salt, sophoridine ferrocenecarboxylate double salt according to claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that, the pharmaceutical composition is prepared into a solid oral preparation, an injection preparation, an emulsion, a cream, a tincture or an ointment.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; the excipients include one or a mixture of a filler, a binder, a disintegrant, a lubricant, a flavoring agent, an enteric coating or a sustained release material.

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