A kind of berberine chinensis acid double salt, preparation method and use thereof

Abstract

The present invention relates to a berberine serrata acid double salt represented by formula (I) and a composition, preparation method and use thereof. The berberine serrata acid double salt represented by formula (I) has anti-tumor / cancer activity.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a berberine chinensis acid double salt, a preparation method thereof and uses thereof. Background Art

[0002] Cancer has become the leading cause of death worldwide.

[0003] Cancer can occur at any age in various organs and tissues. The main types of cancer that lead to death are: lung cancer, colorectal cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, thyroid cancer, pancreatic cancer, prostate cancer, and cervical cancer. Tumors with extremely high morbidity and mortality rates have seriously affected human health and survival. Although some small molecule anticancer drugs have been used in clinical practice, some compounds are undergoing preclinical research. However, most cancer patients are already in the middle to late stages when they discover their condition, and the overall clinical treatment effect is poor, especially the continuous emergence of multidrug resistance, which makes cancer treatment difficult. Therefore, it is urgent to develop new anticancer drugs with high activity and low side effects to meet clinical needs. In particular, lead compounds with anti-tumor / cancer activity are discovered from traditional Chinese medicines, and then they are designed and synthesized to prepare anti-tumor / cancer drugs with high activity and low side effects.

[0004] Berberine, also known as berberine, is an isoquinoline alkaloid isolated from the traditional Chinese Ranunculaceae plant Coptis chinensis Franch. It has long been used as an over-the-counter drug in the clinic to treat intestinal bacterial infections and diarrhea. Modern pharmacological studies have shown that berberine also has good effects in the treatment of cardiovascular and metabolic diseases. Wei Jing, an endocrinologist at Nanjing First Hospital, discovered that berberine has the effects of lowering blood sugar and blood lipids, and revealed the secret of berberine's blood lipid-lowering effect at the molecular level. This study has unearthed new effects of berberine and discovered a blood lipid-lowering Chinese medicine with independent intellectual property rights in my country.

[0005] In addition, berberine also has good therapeutic effects in the treatment of tumors, especially as an auxiliary drug for clinical tumor treatment [(1) Pharm Biol, 2019, 57: 193-225; (2) Front Pharmacol, 2021, 12: e631100; (3) Front Pharmacol, 2020, 10: e1461]. Currently, multiple clinical studies on the anti-cancer effects of berberine are being carried out in an orderly manner (Acta Pharmaceutica Sinica, 2022, 57 (2): 343-352). These examples have shown that berberine has good anti-tumor reproducibility and transformation potential, and berberine can be transformed as a potential drug or lead compound for the auxiliary treatment of tumors.

[0006] Artemisia Rupestris L. is mainly distributed in Xinjiang, China, Central Asia, Europe and other places. It has a long history of folk medicine in Xinjiang. It is clinically used to treat indigestion, abdominal and stomach pain, hepatitis, snake bites, tumors, colds and fevers. Rupestonic acid is one of the characteristic compounds in Artemisia Rupestris L. It is a multifunctional sesquiterpenoid compound, which usually has a broad spectrum of pharmacological activities. Summary of the invention

[0007] The invention prepares a berberine chinensis acid double salt, and the results show that the double salt has strong inhibitory activity on tumor cell lines.

[0008] The present invention provides a berberine chinensis acid double salt represented by formula (I),

[0009]

[0010] The present invention also provides a method for preparing the berberine chinensis double salt represented by the above formula (I), comprising the following steps:

[0011] The artemisinic acid and berberine react to obtain the artemisinic acid berberine complex salt represented by the above formula (I).

[0012] According to an embodiment of the present invention, the reaction temperature is room temperature;

[0013] and / or, the reaction is carried out in an organic solvent, preferably a single solvent or a mixed solvent such as methanol, ethanol, chloroform, tetrahydrofuran, toluene, dichloromethane, ethyl acetate, etc., which can dissolve the two compounds, such as methanol;

[0014] And / or, the molar ratio of artemisinic acid to berberine is 1:(0.8-2), preferably 1:1.

[0015] According to an embodiment of the present invention, the preparation method comprises the following steps:

[0016] 1) reacting artemisinic acid with a base to form a salt, wherein the base is preferably a strong base, more preferably an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide, or lithium hydroxide;

[0017] 2) adding berberine to obtain the berberine ester complex salt represented by the above formula (I).

[0018] According to an embodiment of the present invention, the preparation route is:

[0019]

[0020] According to an embodiment of the present invention, the preparation method comprises the following steps:

[0021] Artemisia ketoacid and sodium hydroxide are mixed in a molar ratio of 1:1, and an appropriate amount of organic solvent is added. The mixture is stirred at room temperature or ultrasonically reacted under nitrogen protection. After TLC detection shows that the artemisia ketoacid is completely salified, an equimolar amount of berberine is added to the reaction system, and the mixture is stirred at 50-80°C under nitrogen protection. After the reaction is completed under TLC monitoring, the reaction solution is concentrated under reduced pressure, the solid is washed 2-3 times with refrigerated deionized water, and the solid is vacuum dried at 40°C to obtain the artemisia ketoacid berberine complex salt shown in the above formula (I).

[0022] The present invention also provides the use of the berberine chinensis acid complex salt represented by the above formula (I) in the preparation of drugs for treating and / or preventing tumors / cancers.

[0023] According to an embodiment of the present invention, the tumor / cancer is selected from breast cancer, lung adenocarcinoma, cervical cancer, preferably breast cancer.

[0024] The present invention also provides a pharmaceutical composition comprising an effective amount of the berberine chinensis acid complex salt represented by the above formula (I).

[0025] According to an embodiment of the present invention, the pharmaceutical composition is used for treating and / or preventing tumors / cancers.

[0026] According to an embodiment of the present invention, the tumor / cancer is selected from breast cancer, lung adenocarcinoma, cervical cancer, preferably breast cancer.

[0027] According to an embodiment of the present invention, the pharmaceutical composition may further include one or more additional therapeutic agents.

[0028] According to an embodiment of the present invention, the pharmaceutical composition may be a solid oral preparation or an injection preparation.

[0029] According to an embodiment of the present invention, the solid oral preparation may be a capsule, a tablet, a granule, a sustained-release preparation, or the like.

[0030] According to an embodiment of the present invention, the injection preparation can be a small needle injection or a freeze-dried injection.

[0031] 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 fillers, binders, disintegrants, lubricants, flavoring agents, enteric coatings or sustained-release materials.

[0032] According to an embodiment of the present invention, the filler comprises: one or a mixture of lactose, sucrose, dextrin, starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium carbonate, and microcrystalline cellulose;

[0033] According to an embodiment of the present invention, the binder comprises one or a mixture of sucrose, starch, sodium carboxymethyl cellulose, methyl cellulose, polyethylene glycol, and water;

[0034] According to an embodiment of the present invention, the disintegrant comprises one or a mixture of starch, cross-linked polymicronizer, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, and sodium carboxymethyl cellulose;

[0035] According to an embodiment of the present invention, the lubricant includes one or a mixture of talc, magnesium stearate, stearic acid, micro powder silica gel, polyethylene glycol-4000, polyethylene glycol-6000;

[0036] According to an embodiment of the present invention, the flavoring agent includes one or a mixture of sucrose, sorbitol, saccharin sodium, maltitol, steviol glycosides, and aspartame;

[0037] According to an embodiment of the present invention, the sustained-release material includes one or a mixture of several of hypromellose and guar gum with different specifications and viscosities.

[0038] 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: 0.01-0.5 parts of berberine bisporus acid complex salt shown in formula (I), 2-20 parts of lactose, 1-10 parts of pregelatinized starch, 1-5 parts of microcrystalline cellulose, 0.1-5 parts of sodium starch glycolate, 2-10 parts of 10% povidone solution, 1-5 parts of magnesium stearate and 1-5 parts of talc.

[0039] According to an embodiment of the present invention, when the pharmaceutical composition is a solid oral preparation, the preparation method thereof comprises:

[0040] 1) After mixing the auxiliary materials, pass through a 40-100 mesh sieve;

[0041] 2) adding the berberine chinensis acid double salt represented by formula (I) to 10% povidone solution and mixing;

[0042] 3) After being evenly mixed in step 2), the mixture is added to the sieved auxiliary materials, ground and mixed, dried at 50-60° C. for 2-3 hours, and tableted.

[0043] The present invention also provides a method for treating tumors / cancers, comprising administering to a patient an effective amount of the artemisinic acid berberine complex salt shown in formula (I) for prevention or treatment.

[0044] According to an embodiment of the present invention, the patient is a human.

[0045] Beneficial Effects

[0046] The present invention provides a berberine sclerotium acid double salt represented by formula (I), a preparation method thereof and a use thereof. The berberine sclerotium acid double salt represented by formula (I) prepared by the present invention has strong inhibitory activity against tumor cell lines. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0048] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0049] Instruments and reagents:

[0050] Artemisinic acid (98%, provided by Researcher Aji Aikebai Isa of Xinjiang Institute of Physical and Chemical Technology, Chinese Academy of Sciences), berberine (98%, Bid Pharmaceuticals); XT4 microscopic melting point meter (Shanghai Jinghe Analytical Instrument Co., Ltd.).

[0051] Example 1: Preparation of berberine tert-butyl ester complex salt

[0052]

[0053] 0.124g (0.5mmol) of artemisinic acid and 0.02g (0.5mmol) of sodium hydroxide were added to a 25mL round-bottom flask, and 5mL of anhydrous methanol was added. The reaction was stirred under nitrogen protection. After TLC detected that artemisinic acid was completely formed into sodium salt, 0.168g (0.5mmol) of berberine was slowly added to the reaction system, and the reaction was stirred under reflux under nitrogen protection. After the reaction was completed by TLC detection, it was concentrated under reduced pressure, the solid was washed with refrigerated deionized water 2-3 times, and the solid was vacuum dried at 40°C to obtain artemisinic acid berberine complex salt, a brown solid. The color state and melting point of the raw materials and products are shown in Table 1.

[0054] Table 1 Color and melting point of raw materials and products

[0055] Physical properties and appearance Artemisinic acid Berberine Berberine chinensis acid double salt color Colorless solid Turmeric solid Brown solid Melting point / ℃ 132-134 204-206 151-153

[0056] Example 2: In vitro anti-tumor activity test

[0057] The compound artemisinic acid berberine complex salt in Example 1 was tested for in vitro antitumor activity using the CCK-8 method. The in vitro inhibitory activity of the compound artemisinic acid berberine complex salt on breast cancer cell lines (MCF-7), lung adenocarcinoma cell lines (A549) and cervical cancer cell lines (Hela) was mainly studied. Breast cancer cell lines (MCF-7), lung adenocarcinoma cell lines (A549) and cervical cancer cell lines (Hela) are derived from cell lines preserved by Ningxia Medical University. The specific test process is described using the test process of breast cancer MCF-7 cell lines as an example:

[0058] (1) Cultivation of breast cancer cell line (MCF-7) and inhibitory activity test process

[0059] The breast cancer cell line MCF-7 was placed in an incubator at 37°C, saturated humidity, and 5% CO2 for 24 hours. When the cells were in the logarithmic growth phase, the upper culture medium was discarded, and digested with 0.25% trypsin-EDTA solution, and then digested with high-glucose medium. The cells were inoculated in a 96-well plate so that the cell density was 5000 cells / well. The 96-well plate was placed in an incubator and cultured for 24 hours. The cell culture medium in the 96-well plate was then discarded. 100 μL of high-glucose medium was added to the 96-well plate, and then 1 μL of the test sample of different concentrations was added to each well (5 replicates were set for each concentration), and then placed in an incubator at 37°C, saturated humidity, and 5% CO2 for continued culture for 48 hours, and then 10 μL of CCK8 was added to each well. After continuing to incubate in a 37°C incubator for 1-4 hours, the absorbance value of each well at a wavelength of 450 nm was measured 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. Negative control was V 高糖培养基 / V DMSO =10:1 mixed solution, with artemisinic acid and berberine chloride as positive controls.

[0060] (2) Cultivation and inhibitory activity testing process of lung cancer cell line (A549) and cervical cancer cell line (Hela)

[0061] The experimental process of inhibiting lung cancer cell line A549 and cervical cancer cell line (Hela) is the same as the screening process of breast cancer cell line (MCF-7).

[0062] The test results show that berberine chinensis acid complex salt has strong inhibitory activity against three tumor cell lines, among which the inhibitory activity against breast cancer cell line (MCF-7) is more prominent. The inhibitory results against breast cancer cell line (MCF-7) are shown in Table 2 below.

[0063] Table 2 The inhibitory effects of berberine chinensis acid complex salt on breast cancer cell lines

[0064] Compound <![CDATA[Inhibitory rate / IC 50 (μM) against breast cancer cell line (MCF-7)]]> Artemisinic acid 1712.0 Berberine 59.1 Berberine chinensis acid double salt 66.6

[0065] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A berberine chinensis acid double salt represented by formula (I), 2. A method for preparing the berberine chinensis double salt of formula (I) according to claim 1, the method comprising the following steps: The artemisinic acid and berberine react to obtain the artemisinic acid berberine complex salt represented by the formula (I).

3. The preparation method according to claim 2, characterized in that: The reaction temperature is room temperature; and / or, the reaction is carried out in an organic solvent; And / or, the molar ratio of artemisinic acid to berberine is 1:(0.8-2).

4. The preparation method according to claim 3, characterized in that: The organic solvent is selected from a single solvent or a mixed solvent of methanol, ethanol, chloroform, tetrahydrofuran, toluene, dichloromethane, and ethyl acetate; And / or, the molar ratio of artemisinic acid to berberine is 1:

1.

5. The preparation method according to claim 2 or 3, characterized in that: The preparation method comprises the following steps: 1) reacting artemisinic acid with a base to form a salt; 2) adding berberine to obtain the berberine ester complex salt represented by formula (I).

6. The preparation method according to claim 5, characterized in that: The base is a strong base.

7. Use of the berberine chinensis acid complex salt represented by formula (I) according to claim 1 in the preparation of a drug for treating and / or preventing tumors / cancers; the tumor / cancer is breast cancer.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the berberine chinensis double salt represented by formula (I) according to claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is used for treating and / or preventing tumors / cancers; the tumor / cancer is breast cancer.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that The pharmaceutical composition further comprises one or more additional therapeutic agents; And / or, the pharmaceutical composition is a solid oral preparation or an injectable preparation; And / or, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition according to claim 10, characterized in that The solid oral preparations are capsules, tablets, granules, and sustained-release preparations, and the injection preparations are small-needle injections and freeze-dried injections.

12. The pharmaceutical composition according to claim 10, characterized in that The auxiliary materials include one or a mixture of fillers, binders, disintegrants, lubricants, flavoring agents, enteric coatings or sustained-release materials.

13. The pharmaceutical composition according to claim 12, characterized in that The filler includes: one or a mixture of lactose, sucrose, dextrin, starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium carbonate, and microcrystalline cellulose; and / or, the binder comprises one or a mixture of sucrose, starch, sodium carboxymethyl cellulose, methyl cellulose, polyethylene glycol, and water; And / or, the disintegrant comprises one or a mixture of starch, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose; And / or, the lubricant includes one or a mixture of talc, magnesium stearate, stearic acid, micro-powder silica gel, polyethylene glycol-4000, polyethylene glycol-6000; And / or, the flavoring agent includes one or a mixture of sucrose, sorbitol, saccharin sodium, maltitol, steviol glycosides, and aspartame; And / or, the sustained-release material includes one or a mixture of several of hydroxypropyl methylcellulose and guar gum with different viscosity specifications.

14. The pharmaceutical composition according to claim 10, characterized in that When the pharmaceutical composition is a solid oral preparation, it comprises the following components in parts by weight: 0.01-0.5 parts of berberine bisporus acid complex salt shown in formula (I), 2-20 parts of lactose, 1-10 parts of pregelatinized starch, 1-5 parts of microcrystalline cellulose, 0.1-5 parts of sodium starch glycolate, 2-10 parts of 10% povidone solution, 1-5 parts of magnesium stearate and 1-5 parts of talc.

15. The pharmaceutical composition according to claim 10, characterized in that When the pharmaceutical composition is a solid oral preparation, the preparation method thereof comprises: 1) After mixing the auxiliary materials, pass through a 40-100 mesh sieve; 2) adding the berberine chinensis acid double salt represented by formula (I) to 10% povidone solution and mixing; 3) After being evenly mixed in step 2), the mixture is added to the sieved auxiliary materials, ground and mixed, dried at 50-60° C. for 2-3 hours, and tableted.

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