CN116082147A - Rupestonic acid berberine double salt, preparation method and application thereof - Google Patents

Abstract

The invention relates to a rupestonic acid berberine double salt shown in a formula (I), a composition, a preparation method and application thereof. The rupestonic acid berberine double salt shown in the formula (I) has anti-tumor/cancer activity.

Description

Rupestonic acid berberine double salt, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a rupestonic acid berberine double salt, a preparation method and application thereof.

Background

Cancer has become the leading fatal disease worldwide.

Cancers can occur in various organs and tissues at any age, with the major types of cancer leading to death: 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 have severely affected human health and survival. While some small molecule anticancer drugs have been used clinically, some compounds are being studied preclinically. However, most cancer patients find the disease state to be middle to late stage, the overall clinical treatment effect is poor, and especially the continuous occurrence of multi-drug resistance makes the treatment of cancer difficult and serious. Therefore, development of a novel anticancer drug with high activity and low side effect is urgent to meet clinical demands. Especially, the lead compound with anti-tumor/cancer activity is found from the traditional Chinese medicine, and then the lead compound is designed and synthesized to prepare the anti-tumor/cancer medicine with high activity and low side effect.

Berberine, also known as berberine. Is an isoquinoline alkaloid separated from traditional Ranunculaceae plant Coptis chinensis Franch. Clinically, the medicine is used for treating intestinal bacterial infection and diarrhea for a long time as an over-the-counter medicine. Modern pharmacological studies have shown that: berberine also has good effect in treating cardiovascular diseases and metabolic diseases. The first hospital endocrinology expert Wei Jing in Nanjing finds that berberine has the efficacy of reducing blood sugar and blood fat, and reveals the berberine's mysterious for reducing blood fat from the molecular level. The research discovers new efficacy of berberine and discovers the hypolipidemic traditional Chinese medicine with independent intellectual property rights in China.

In addition, berberine has good curative effect in the aspect of treating tumors, and can be especially used 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, several clinical studies on berberine anticancer effects are ongoing in order (Acta Pharmaceutica Sinica,2022,57 (2): 343-352). These examples have shown that berberine has good antitumor reproducibility and transformation potential, and berberine can be modified as a potential drug or lead compound for adjuvant treatment of tumors.

Artemisia rupestris (Artemisia Rupestris L.) is mainly distributed in Xinjiang, zhongya, europe and the like. The medicine taking history is long in Xinjiang people. Can be used for treating dyspepsia, gastralgia, hepatitis, snake bite, tumor, common cold, and fever. Rupestonic acid (rupestonic acid) is one of the characteristic compounds in alpine yarrow herb of Xinjiang, is a sesquiterpenoid compound containing a multifunctional group, and the sesquiterpenoid compound generally has broad-spectrum pharmacological activity.

Disclosure of Invention

The invention prepares the rupestonic acid berberine double salt, and the result shows that the double salt has stronger inhibition activity on tumor cell strains.

The invention provides a compound salt of rupestonic acid berberine shown in a formula (I),

the invention also provides a preparation method of the rupestonic acid berberine double salt shown in the formula (I), which comprises the following steps:

reacting rupestonic acid with berberine to obtain rupestonic acid berberine double salt shown in the formula (I).

According to an embodiment of the invention, the reaction temperature is room temperature;

and/or the reaction is carried out in an organic solvent, preferably: a single solvent or a mixed solvent capable of dissolving both compounds, such as methanol, ethanol, chloroform, tetrahydrofuran, toluene, methylene chloride, ethyl acetate, etc.;

and/or, the molar ratio of rupestonic acid to berberine is 1 (0.8-2), preferably 1:1.

According to an embodiment of the invention, the preparation method comprises the following steps:

1) The rupestonic acid reacts with a base to form a salt, the base preferably being a strong base, more preferably an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide, lithium hydroxide;

2) Adding berberine to obtain berberine compound salt of rupestonic acid shown in formula (I).

According to an embodiment of the invention, the preparation route is:

according to an embodiment of the invention, the preparation method comprises the following steps:

mixing rupestonic acid and sodium hydroxide according to a molar ratio of 1:1, adding a proper amount of organic solvent, stirring at room temperature under nitrogen protection for reaction or performing ultrasonic reaction, adding berberine with an equal molar amount into a reaction system after TLC detection of complete salification of rupestonic acid, stirring at 50-80 ℃ under nitrogen protection for reaction, concentrating reaction liquid under reduced pressure after TLC monitoring reaction is completed, washing solid with refrigerated deionized water for 2-3 times, and vacuum drying solid at 40 ℃ to obtain the rupestonic acid berberine double salt shown in the formula (I).

The invention also provides application of the rupestonic acid berberine double salt shown in the formula (I) in preparing medicines for treating and/or preventing tumors/cancers.

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

The invention also provides a pharmaceutical composition which comprises an effective amount of the rupestonic acid berberine double salt shown in the formula (I).

According to an embodiment of the invention, the pharmaceutical composition is for the treatment and/or prevention of tumors/cancers.

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

According to embodiments of the present invention, the pharmaceutical composition may further comprise one or more additional therapeutic agents.

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

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

According to an embodiment of the present invention, the injection preparation may be a small needle injection, a freeze-dried injection.

According to an embodiment of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; the auxiliary materials comprise one or a mixture of more of filler, adhesive, disintegrating agent, lubricant, flavoring agent, enteric coating or slow release material.

According to an embodiment of the present invention, the filler includes: lactose, sucrose, dextrin, starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose or their mixture;

according to an embodiment of the invention, the binder comprises one or a mixture of several of sucrose, starch, sodium carboxymethyl cellulose, methyl cellulose, polyethylene glycol and water;

according to an embodiment of the present invention, the disintegrating agent comprises one or a mixture of several of starch, cross-linked polyketone, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose;

according to the embodiment of the invention, the lubricant comprises one or a mixture of more of talcum powder, magnesium stearate, stearic acid, micro powder silica gel, polyethylene glycol-4000 and polyethylene glycol-6000;

according to an embodiment of the invention, the flavoring agent comprises one or a mixture of several of sucrose, sorbitol, saccharin sodium, maltitol, steviol glycoside and aspartame;

according to the embodiment of the invention, the slow release material comprises one or a mixture of a plurality of hydroxypropyl methylcellulose and guar gum with different specification viscosities.

According to an embodiment of the invention, when the pharmaceutical composition is a solid oral preparation, the pharmaceutical composition comprises the following components in parts by weight: 0.01-0.5 part of rupestonic acid berberine double 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 carboxymethyl starch sodium, 2-10 parts of 10% povidone solution, 1-5 parts of magnesium stearate and 1-5 parts of talcum powder.

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

1) Mixing the auxiliary materials, and sieving with a 40-100 mesh sieve;

2) Adding a double salt of rupestonic acid berberine shown in the formula (I) into a 10% povidone solution, and mixing;

3) And 2) uniformly mixing in the step 2), adding the mixture into the sieved auxiliary materials, grinding and mixing, drying at 50-60 ℃ for 2-3 hours, and tabletting to obtain the finished product.

The invention also provides a method of treating a tumor/cancer comprising administering to a patient a prophylactically or therapeutically effective amount of a complex salt of rupestonic acid berberine of formula (I).

According to an embodiment of the invention, the patient is a human.

Advantageous effects

The invention provides a rupestonic acid berberine double salt shown in a formula (I), a preparation method and application thereof. The rupestonic acid berberine double salt shown in the formula (I) prepared by the invention has strong inhibition activity on tumor cell lines.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

Instrument and reagent:

rupestonic acid (98%, provided by the institute of physical and chemical technology, xinjiang, china, a Ji Aike Bayer Ai Sa researcher), berberine (98%, piobtained pharmaceutical); XT4 microscopy melting point tester (vitex marigold and analytical instruments limited).

Example 1: preparation of rupestonic acid berberine double salt

0.124g (0.5 mmol) of rupestonic acid and 0.02g (0.5 mmol) of sodium hydroxide are added into a 25mL round bottom flask, 5mL of absolute methanol is added, stirring reaction is carried out under nitrogen protection, after TLC detects that the rupestonic acid completely forms sodium salt, 0.168g (0.5 mmol) of berberine is slowly added into the reaction system, and reflux stirring reaction is carried out under nitrogen protection. After the TLC detection reaction is finished, concentrating under reduced pressure, washing the solid with refrigerated deionized water for 2-3 times, and vacuum drying the solid at 40 ℃ to obtain the rupestonic acid berberine double salt and brown solid. The color states and melting points of the raw materials and the products are shown in Table 1.

TABLE 1 color of raw materials and products and melting point

Physical constant and appearance	Rupestonic acid	Berberine	Rupestonic acid berberine double salt
				Color of	Colorless solid	Ginger yellow solid	Brown solid
Melting Point/. Degree.C	132-134	204-206	151-153

Example 2: in vitro antitumor Activity test

An in vitro anti-tumor activity test was performed on the compound rupestonic acid berberine double salt in example 1 by using a CCK-8 method. The in vitro inhibitory activity of the recombinant strain against the breast cancer cell line (MCF-7), lung adenocarcinoma cell line (A549) and cervical cancer cell line (Hela) was mainly studied. The breast cancer cell line (MCF-7), lung adenocarcinoma cell line (A549) and cervical cancer cell line (Hela) are derived from cell lines stored in Ningxia university of medical science. The specific test procedure is described by taking the test procedure of the breast cancer MCF-7 cell strain as an example:

(1) Culture of breast cancer cell line (MCF-7) and inhibitory activity testing procedure

Placing breast cancer cell strain MCF-7 at 37deg.C, saturated humidity, and containing 5% CO ₂ The culture was carried out in an incubator for 24 hours, and when the cells were in the logarithmic growth phase, the upper layer culture broth was aspirated, and after digestion with a 0.25% trypsin-EDTA solution, the digestion was stopped using a high sugar medium. And cells were seeded in 96-well plates such that the cell density was 5000 cells/well. The 96-well plate was placed in an incubator for 24 hours. With consequent pipetting of the cell culture broth in the 96-well plate. And 100. Mu.L of high sugar culture medium is added into a 96-well plate, then 1. Mu.L of test samples with different concentrations (5 multiple wells are arranged for each concentration) are added into each well, and then the mixture is placed at 37 ℃ and saturated humidity with 5% CO ₂ After 48h of continuous incubation in an incubator of (2) 10. Mu.L of CCK8 was added to each well, and after further incubation in the incubator at 37℃for 1-4h, the absorbance value of each well at a wavelength of 450nm was measured on a multifunctional microplate reader. According to the inhibition ratio = [ (OD) _{Control cells} -OD _{Medicated cell} )/(OD _{Control cells} -OD _{Blank space} )]X 100. Negative control is V _{High sugar culture medium} /V _DMSO Mixed solution=10:1 with rupestonic acid and berberine chloride as positive control.

(2) Culture of lung cancer cell line (A549) and cervical cancer cell line (Hela) and inhibitory activity test procedure

The experimental procedure for inhibiting lung cancer cell line A549 and cervical cancer cell line (Hela) was the same as the screening procedure for breast cancer cell line (MCF-7).

The test results show that: the rupestonic acid berberine double salt has strong inhibition activity on three tumor cell lines, wherein the inhibition activity on breast cancer cell line (MCF-7) is more prominent. The results of inhibition of breast cancer cell line (MCF-7) are shown in Table 2 below.

TABLE 2 inhibition of Brevibacterium keto acid berberine double salt on breast cancer cell lines

Compounds of formula (I)	Inhibition rate/IC for breast cancer cell line (MCF-7) 50 (μM)
		Rupestonic acid	1712.0
Berberine	59.1
		Rupestonic acid berberine double salt	66.6

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A compound salt of rupestonic acid berberine shown in formula (I),

2. the preparation method of the berberine compound salt of rupestonic acid shown in the formula (I) in claim 1 comprises the following steps:

reacting rupestonic acid with berberine to obtain rupestonic acid berberine double salt shown in the formula (I).

3. The method according to claim 2, wherein,

the reaction temperature is room temperature;

and/or the reaction is carried out in an organic solvent, preferably: a single solvent or a mixed solvent capable of dissolving both compounds, such as methanol, ethanol, chloroform, tetrahydrofuran, toluene, methylene chloride, ethyl acetate, etc.;

and/or, the molar ratio of rupestonic acid to berberine is 1 (0.8-2), preferably 1:1.

4. A method of preparation according to claim 2 or 3, characterized in that the method of preparation comprises the steps of:

1) The rupestonic acid reacts with a base to form a salt, the base preferably being a strong base, more preferably an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide, lithium hydroxide;

2) Adding berberine to obtain berberine compound salt of rupestonic acid shown in formula (I);

for example: mixing rupestonic acid and sodium hydroxide according to a molar ratio of 1:1, adding a proper amount of organic solvent, stirring at room temperature under nitrogen protection for reaction or performing ultrasonic reaction, adding berberine with an equal molar amount into a reaction system after TLC detection of complete salification of rupestonic acid, stirring for reaction at 50-80 ℃ under nitrogen protection, concentrating reaction liquid under reduced pressure after TLC monitoring reaction is completed, washing solid with refrigerated deionized water for 2-3 times, and vacuum drying solid at 40 ℃ to obtain the rupestonic acid berberine double salt shown in the formula (I).

5. Use of a complex salt of berberine of rupestonic acid of formula (I) according to claim 1 for the preparation of a medicament for the treatment and/or prevention of tumors/cancers;

preferably, the tumor/cancer is selected from breast cancer, lung adenocarcinoma, cervical cancer, e.g. breast cancer.

6. A pharmaceutical composition comprising an effective amount of a complex salt of rupestonic acid berberine of formula (I) according to claim 1.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is for use in the treatment and/or prevention of tumors/cancers;

and/or the tumor/cancer is selected from breast cancer, lung adenocarcinoma, cervical cancer, preferably breast cancer.

8. The pharmaceutical composition according to claim 6 or 7, wherein,

the pharmaceutical composition may further comprise one or more additional therapeutic agents;

and/or the pharmaceutical composition can be a solid oral preparation or an injection preparation, wherein the solid oral preparation is preferably a capsule, a tablet, a granule, a slow release agent and the like, and the injection preparation is preferably a small needle injection or a freeze-dried injection;

and/or, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; preferably, the auxiliary materials comprise one or a mixture of more of a filler, an adhesive, a disintegrating agent, a lubricant, a flavoring agent, an enteric coating or a slow release material; preferably, the filler comprises: lactose, sucrose, dextrin, starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose or their mixture; preferably, the adhesive comprises one or a mixture of more than one of sucrose, starch, sodium carboxymethyl cellulose, methyl cellulose, polyethylene glycol and water; preferably, the disintegrating agent comprises one or a mixture of more of starch, cross-linked polyketone, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and sodium carboxymethyl cellulose; preferably, the lubricant comprises one or a mixture of more of talcum powder, magnesium stearate, stearic acid, micro powder silica gel, polyethylene glycol-4000 and polyethylene glycol-6000; preferably, the flavoring agent comprises one or a mixture of more than one of sucrose, sorbitol, saccharin sodium, maltitol, stevioside and aspartame; preferably, the slow release material comprises one or a mixture of a plurality of hydroxypropyl methylcellulose and guar gum with different specification viscosities;

and/or, when the pharmaceutical composition is a solid oral preparation, the pharmaceutical composition comprises the following components in parts by weight: 0.01-0.5 part of rupestonic acid berberine double 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 carboxymethyl starch sodium, 2-10 parts of 10% povidone solution, 1-5 parts of magnesium stearate and 1-5 parts of talcum powder.

And/or, when the pharmaceutical composition is a solid oral preparation, the preparation method comprises the following steps:

1) Mixing the auxiliary materials, and sieving with a 40-100 mesh sieve;

2) Adding a double salt of rupestonic acid berberine shown in the formula (I) into a 10% povidone solution, and mixing;

3) And 2) uniformly mixing in the step 2), adding the mixture into the sieved auxiliary materials, grinding and mixing, drying at 50-60 ℃ for 2-3 hours, and tabletting to obtain the finished product.