Use of c-methylated high isoflavone compounds in the preparation of antitumor drugs

Abstract

The application relates to application of C-methylated high isoflavone compounds in preparation of antitumor drugs and belongs to the technical field of drug application. In order to solve the problem that the existing antitumor activity is not much reported, the application of C-methylated high isoflavone compounds in preparation of antitumor drugs is provided, the C-methylated high isoflavone compounds are selected from compounds shown in the following formula 1 or formula 2: the C-methylated high isoflavone compounds or pharmaceutically acceptable salts thereof are used as active ingredients for preparation of antitumor drugs for preventing and / or treating adrenal pheochromocytoma (PC-12) and / or human brain astrocytoma (U-87MG). The C-methylated high isoflavone compounds have the advantages of high activity and high inhibition capacity, can be extracted from polygonatum sibiricum and have good drug safety.

Description

Technical Field

[0001] This invention relates to the application of C-methylated isoflavone compounds in the preparation of antitumor drugs, and belongs to the technical field of pharmaceutical applications. Background Technology

[0002] Cancer is a disease with a complex pathogenesis and has become a major threat to human health, with its incidence and mortality rates rising year by year. While my country's cancer incidence rate is close to the world average, its mortality rate is higher. With increasing age, both the incidence and mortality rates for men and women in China gradually increase. Finding and discovering new drugs for the treatment and prevention of cancer is a major current challenge. Natural products are considered an important source of novel chemical entities with unique pharmacological activities and have long been regarded as a valuable source for drug design, especially in the development of anti-tumor drugs. Therefore, finding novel anti-tumor drugs from natural products has significant research value. my country has abundant traditional Chinese medicine resources, which have shown good effects in treating malignant tumors, whether in alleviating clinical symptoms, improving quality of life, preventing recurrence and metastasis, prolonging survival, or in combining with radiotherapy and chemotherapy to enhance efficacy and reduce toxicity.

[0003] PC-12 cells are a cell line derived from pheochromocytoma clones, commonly obtained from calves, rats, cats, and pigs. They secrete catecholamine hormones such as adrenaline and noradrenaline, and also possess characteristics of nerve cells, including the presence of various voltage-dependent sodium, potassium, and calcium ion channels and receptor activation channels. A key characteristic of PC-12 cells is their ability to inhibit cell proliferation and promote neurite growth, thus making them widely used in nervous system research. U-87MG cells are human glioblastoma cells, derived from gliomas, and can form tumors when implanted subcutaneously in nude mice. Since their initial establishment at Uppsala University in Sweden in 1966, these cells have been widely used as common cells for studying human gliomas. The search for and development of antitumor compounds targeting these two types of tumor cells is of significant research importance.

[0004] On the other hand, the traditional Chinese medicine Polygonatum is the dried rhizome of *P. kingianum* Coll. et Hemsl., *P. sibiricum* Red., or *P. cyrtonema* Hua, all belonging to the genus *P.* of the Liliaceae family. It is a commonly used Chinese medicine listed in the Chinese Pharmacopoeia and has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. *P. cyrtonema* Hua, also known as ginger-shaped Polygonatum, is derived from the plant *P. cyrtonema* and is mainly produced in southern my country. It has the effects of tonifying the kidneys and replenishing essence, nourishing yin and moistening dryness, and has long been used to treat kidney deficiency and spleen and stomach weakness. Currently, compounds extracted from it have been found to have anti-tumor functions against gastric cancer and colon cancer, but there is still a lack of information on its activity in other tumors.

[0005] Existing literature discloses high-isoflavonoid compounds and their uses. These include three novel high-isoflavonoid compounds isolated from Polygonatum odoratum, which exhibit good antitumor activity. Their structural formulas are shown below:

[0006]

[0007] However, the compounds disclosed in this literature mainly exhibit good antitumor activity against leukemia, colon cancer, lung cancer, sarcoma, or liver cancer. However, there are many types of tumor cells, and these compounds have certain activity against the aforementioned tumors. There are currently no further discoveries regarding new applications of these compounds, and no literature reports their activity against the aforementioned PC-12 and U-87MG tumor cells. Furthermore, different substituents can produce different performance characteristics. Summary of the Invention

[0008] To address the deficiencies in the existing technologies, this invention provides an application of C-methylated high isoflavone compounds in the preparation of antitumor drugs. The problem solved is to provide new C-methylated high isoflavone compounds that exhibit high antitumor activity against pheochromocytoma (PC-12) and human astrocytoma cells (U-87MG).

[0009] The objective of this invention is achieved through the following technical solution: the application of a C-methylated high isoflavone compound in the preparation of antitumor drugs, wherein the C-methylated high isoflavone compound is selected from compounds with the following structural formulas: Formula 1 or Formula 2.

[0010]

[0011] Characterized in that the C-methylated isoflavone compound or its pharmaceutically acceptable salt is used as an active ingredient in the preparation of antitumor drugs for the prevention and / or treatment of pheochromocytoma (PC-12) and / or glioblastoma (U-87MG) of the human brain.

[0012] Through structural analysis and research of C-methylated isoflavone compounds, it was found that different substituents at the 6- or 8-position of the parent nucleus have different effects on their antitumor activity. Extensive research and validation revealed that when the C atoms at the 6- or 8-position of the parent nucleus of C-methylated isoflavone compounds are partially or simultaneously methylated, the methylation structure improves their selectivity and activity against tumor cells, resulting in inhibitory activity against new tumor cells. This leads to superior antitumor activity against pheochromocytoma (PC-12) and / or glioblastoma (U-87MG) in human brain cells. The tumor activity is the first to be discovered, demonstrating the therapeutic and / or preventive functions of the compounds of Formula 1 and Formula 2 in the aforementioned adrenal pheochromocytoma (PC-12) and / or human astrocytoma (U-87MG). They exhibit excellent inhibitory ability against adrenal pheochromocytoma (PC-12) and / or human astrocytoma (U-87MG) cells, thus achieving good anti-tumor activity. Furthermore, both compounds of Formula 1 and Formula 2 can be extracted from plants of the Polygonatum genus, exhibiting good safety. Moreover, their use as active ingredients in anti-tumor drugs to formulate corresponding drugs also shows good market application prospects.

[0013] In the application of the aforementioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the pharmaceutically acceptable salt of the C-methylated isoflavone compounds is selected from the acid salts of the C-methylated isoflavone compounds. More preferably, the acid salt of the C-methylated isoflavone compounds can be an organic acid salt or an inorganic acid salt; that is, the acid used in the acid salt can be an organic acid or an inorganic acid. Inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc., and organic acids include acetic acid, propionic acid, glycolic acid, maleic acid, oxalic acid, toluenesulfonic acid, methanesulfonic acid, fumaric acid, tartaric acid, salicylic acid, etc. This can better improve the solubility and stability of the drug while maintaining its active properties. As a further preferred embodiment, the acid salt of the C-methylated isoflavone compound is selected from the inorganic acid salts of the C-methylated isoflavone compound, specifically from the hydrochloride, hydrobromide, and sulfate salts of the C-methylated isoflavone compound; the organic acid salt of the C-methylated isoflavone compound is selected from the maleate, oxalate, toluenesulfonate, fumarate, or tartrate salts of the C-methylated isoflavone compound. That is, it can be the hydrochloride, hydrobromide, sulfate, maleate, oxalate, toluenesulfonate, fumarate, or tartrate salts of the compound of Formula 1 above; or it can be the hydrochloride, hydrobromide, sulfate, maleate, oxalate, toluenesulfonate, fumarate, or tartrate salts of the compound of Formula 2 above.

[0014] In the application of the aforementioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the drug further includes a pharmaceutically acceptable carrier and / or excipients. Appropriate carriers and excipients can be added according to the needs of the drug being prepared. These excipients may include, but are not limited to, diluents, fillers, binders, wetting agents, disintegrants, and surfactants. The addition of excipients can better form the corresponding dosage form of the antitumor drug. Pharmaceutically acceptable carriers may include solid dispersants.

[0015] In the application of the above-mentioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the dosage form of the drug is selected from capsules, granules, injections, tablets, aerosols, oral liquids or sustained-release formulations.

[0016] In the application of the above-mentioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the C-methylated isoflavone compounds are extracted from Polygonatum odoratum.

[0017] In the application of the above-mentioned C-methylated isoflavone compounds in the preparation of antitumor drugs, the extraction method is preferably as follows:

[0018] A. After drying and pulverizing the rhizomes of Polygonatum multiflorum, extract them with an alcohol solvent aqueous solution. Concentrate the extract to remove the solvent and obtain the concentrate.

[0019] B. Add water to the concentrate for mixing and dispersion, extract with petroleum ether, then extract with ethyl acetate, collect the ethyl acetate phase, elute by column chromatography on a silica gel column, collect the eluent containing compounds of formula 1 and formula 2, and then purify and separate to obtain compounds of formula 1 and formula 2 of C-methylated isoflavone compounds.

[0020] The above-mentioned method directly uses Polygonatum multiflorum as raw material. It first uses an alcohol solvent to extract and remove the alcohol-soluble substances in the early stage, and then uses petroleum ether extraction and ethyl acetate extraction to obtain the ethyl acetate fraction. The ethyl acetate fraction also contains the two active compounds mentioned above, which shows that the corresponding compounds can be effectively extracted from Polygonatum multiflorum. They also have good inhibitory activity against PC12 and U-87MG cells. At the same time, the method of extraction and analysis from plants is easy to operate and has the advantage of simple operation.

[0021] In the application of the above-mentioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the alcohol solvent in step A is selected from methanol, ethanol or propanol.

[0022] In the application of the above-mentioned C-methylated isoflavone compounds in the preparation of antitumor drugs, preferably, the column chromatography in step B is performed using a gradient elution with dichloromethane-ethyl acetate eluent. This method has good elution effect and can more effectively obtain the desired extracts of compounds of formula 1 and formula 2.

[0023] In summary, compared with the prior art, the present invention has the following advantages:

[0024] 1. By screening and methylating the C atom at the 6th or 8th position of the parent nucleus of high isoflavone compounds, or both compounds of formula 1 and formula 2 are obtained. These compounds have the advantages of high activity and high inhibitory ability in the prevention and / or treatment of human brain astroblastoma and / or adrenal medullary pheochromocytoma, and also have good drug safety as they can be extracted from Polygonatum plants.

[0025] 2. Forming the above-mentioned compounds of Formula 1 and Formula 2 into acid salts can better improve the solubility and stability of the drug, while maintaining the drug's activity. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below through specific embodiments, but the present invention is not limited to these embodiments.

[0027] Example 1

[0028] 10 kg of dried and pulverized rhizomes of Polygonatum cyrtonema were extracted with 10 L of 90% methanol aqueous solution at room temperature. The extraction was performed five times, each extraction lasting 24 hours. The extracts from the five extractions were collected and combined. The combined extract was then concentrated under reduced pressure to remove methanol, yielding 2.1 kg of extract. Ethanol or propanol can also be used instead of methanol for extraction.

[0029] After adding 2L of water to the above-obtained extract for dispersion, it was then extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain four components: petroleum ether, ethyl acetate, n-butanol, and water. The ethyl acetate component obtained from the three ethyl acetate extracts was concentrated and the solvent was removed, resulting in a residue of 22.6g.

[0030] The residue was subjected to silica gel column chromatography with a dichloromethane-ethyl acetate gradient elution (dichloromethane to ethyl acetate volume ratio from 10:1 to 0:1, v / v). Based on TLC color development, five fractions Fr.1–Fr.5 were obtained sequentially. The fraction Fr.2 (6.7 g) obtained from the second separation was subjected to MCI column chromatography with a methanol-water gradient elution (methanol to water volume ratio of 50:50-70:30-85:15-100:0, v / v) to obtain seven subfractions Fr.2A–Fr.2G.

[0031] The subfraction Fr.2B was further purified and separated by gel electrophoresis (Sephadex LH-20, MeOH) column chromatography and semi-preparative HPLC (mobile phase: MeCN-H2O 70:30, v / v) to obtain a colorless oily compound of formula 1 (25.1 mg) and a colorless oily compound of formula 2 (1.7 mg). During HPLC purification and separation, the t corresponding to compound 1... R =9.3min, corresponding to the t of compound 2 R =11.2min.

[0032] The structures of the compounds of Formula 1 and Formula 2 obtained above were analyzed and confirmed.

[0033] The NMR spectrum analysis of compound 1 is shown below:

[0034] 1 H-NMR (in CD3OD, 400MHz): δ H4.07(1H,dd,J=11.7,3.2Hz,H-2a),4.22(1H,dd,J=11.7,5.6Hz,H-2b),2.92 (1H,m,H-3),5.90(1H,s,H-8),3.15(1H,dd,J=14.0,4.3Hz,H-9a),2.57(1H,d d,J=14.0,8.9Hz,H-9b),6.36(1H,d,J=2.1Hz,H-3'),6.33(1H,dd,J=7.8,2. 1Hz,H-5'),6.94(1H,d,J=7.8Hz,H-6'),1.91(3H,s,Me-6),3.71(3H,s,OMe).

[0035] 13 C-NMR (in CD3OD, 100MHz): δ C 70.6(C-2),46.2(C-3),200.5(C-4),102.8(C-4a),166.0(C-5),105.5(C-6),163.2(C-7),96.1(C-8),161.6(C-8a),28. 1(C-9),118.1(C-1'),157.5(C-2'),102.3(C-3'),161.1(C-4'),105.5(C-5'),132.6(C-6'),7.5(C-Me),55.6(C-OMe).

[0036] The NMR spectrum analysis of compound 2 is as follows:

[0037] 1 H-NMR (in CD3OD, 400MHz): δ H 4.09(1H,dd,J=11.3,7.7Hz,H-2a),4.24(1H,dd,J=11.3,4.4Hz,H-2b),2.93(1H,m,H-3),3.16(1H,dd,J=13.9,5.1Hz,H-9a),2.59(1H,dd, J=13.9,9.6Hz,H-9b),6.36(1H,s,H-3'),6.33(1H,d,J=8.2Hz,H-5'),6.94(1H,d,J=8.2Hz,H-6'),1.96(3H,s,Me-6,8),3.72(3H,s,OMe).

[0038] 13 C-NMR (in CD3OD, 100MHz): δ C71.1(C-2),47.0(C-3),201.3(C-4),103.3(C-4a),161.1(C-5),105.2(C-6),164.3(C-7),104.2(C-8),159.8(C-8a),28.7(C- 9),118.9(C-1'),158.1(C-2'),103.0(C-3'),161.7(C-4'),106.2(C-5'),133.1(C-6'),8.3(6-Me),7.9(8-Me),56.1(C-OMe).

[0039] Example 2

[0040] The compounds of formula 1 and formula 2 obtained in Example 1 above can be prepared into corresponding acid salts, such as hydrochloride, sulfate, maleate, fumarate, or tartrate salts of compounds of formula 1 or formula 2. During preparation, the obtained compounds of formula 1 or formula 2 are added to the corresponding acid system and stirred thoroughly to obtain the corresponding acid salts of compounds of formula 1 or formula 2, such as hydrochloride, maleate, sulfate, or fumarate salts of compounds of formula 1; or hydrochloride, sulfate, maleate, or fumarate salts of compounds of formula 2.

[0041] Furthermore, compounds of formula 1 and formula 2 are used as active ingredients to formulate antitumor drugs. The amount of compounds of formula 1 and formula 2 used in the antitumor drugs is the effective amount added, and any amount that has inhibitory activity against PC-12 or U-87MG can be used. Further, appropriate carriers, excipients, etc., may be added as needed. These excipients may include, but are not limited to, diluents, fillers, binders, wetting agents, disintegrants, and surfactants.

[0042] The dosage forms of the above-mentioned anti-tumor drugs can be processed into capsules, granules, injections, tablets, aerosols, oral liquids, or sustained-release formulations, depending on the dosage requirements.

[0043] Example 3

[0044] The antitumor activities (PC-12 and U-87MG) of the compounds of Formula 1 and Formula 2 obtained above were determined and analyzed.

[0045] 1. Cell Culture

[0046] The U-87MG and PC-12 cells used were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Alternatively, these U-87MG and PC-12 cells can be purchased through other channels.

[0047] U-87MG cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (Universal Biotech Co., Ltd, Shanghai, China) and 1% penicillin / streptomycin (Sangon Biotech Co., Ltd, Shanghai, China), and then in a 37°C cell culture incubator with 95% air and 5% CO2 (MEMMERT GmbH+Co.KG, Schwabach, Germany). The medium was changed every 2 days.

[0048] PC-12 cells were cultured in RPMI 1640 (11875093) medium supplemented with 10% fetal bovine serum, under the same conditions as the U-87MG cell line.

[0049] 2. Assay of the inhibitory activity of U-87MG and PC-12 cells

[0050] U-87MG and PC-12 cells were cultured in 96-well plates at a rate of 4 × 10⁻⁶. 4 After culturing cells / mL for 24 h, 0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM of the test compound (dissolved in DMSO) and the positive control drug (paclitaxel) were added, and the cells were incubated for another 48 h.

[0051] Then, the cell culture medium was replaced with fresh medium containing 10% (v / v) MTT (5 mg / mL), and the cells were incubated for another 3 hours. Afterward, they were incubated in formaldehyde solvent at 37°C for 1 hour, followed by incubation using a Multimode Microplate Reader (TECANSpark). (Switzerland) Read the absorbance at OD570 and OD690.

[0052] The formula for calculating relative cell viability is:

[0053] Relative cell viability = (OD570s - OD690s) / (OD5700 - OD6900) × 100%, where OD570s and OD690s are the absorbance of the tested sample at 570 nm and 690 nm, respectively, and OD5700 and OD6900 are the absorbance of the negative control at 570 nm and 690 nm, respectively. Data were analyzed using GraphPad Prism version 6.0. Each experiment was performed in quadruplicate, repeated three times. Specific antitumor data are shown in Table 1.

[0054] Table 1 (Inhibitory activity of compounds of formula 1 and formula 2 against PC-12 and U-87MG) a )

[0055]

[0056] In this context, the superscript 'a' represents the average of the three experimental data.

[0057] The superscript b indicates that it was not detected.

[0058] The superscript 'c' indicates a control group.

[0059] As can be seen from the data in Table 1 above, the compounds of Formula 1 and Formula 2 of the present invention exhibit good antitumor activity against PC-12 and U-87MG tumor cells, IC50 50 The activity of both also showed good levels (all less than 10 μM), and the tumor activity of these two substances has not been reported before. This new application was discovered through specific experimental procedures.

[0060] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0061] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

Claims

1. The application of C-methylated isoflavone compounds in the preparation of antitumor drugs, wherein the C-methylated isoflavone compounds are selected from compounds of Formula 1 or Formula 2 as shown below: Its features are, The compound of Formula 1 or a pharmaceutically acceptable salt thereof is used as an active ingredient in the preparation of an antitumor drug for the prevention and / or treatment of pheochromocytoma of the adrenal gland and / or glioblastoma of the human brain. The compound of Formula 2 or a pharmaceutically acceptable salt thereof is used as an active ingredient in the preparation of an antitumor drug for the prevention and / or treatment of pheochromocytoma of the adrenal gland.

2. The application of the C-methylated isoflavone compound according to claim 1 in the preparation of antitumor drugs, characterized in that, The pharmaceutically acceptable salts of the C-methylated isoflavones are selected from the acid salts of the C-methylated isoflavones.

3. The application of the C-methylated isoflavone compound according to claim 2 in the preparation of antitumor drugs, characterized in that, The pharmaceutically acceptable salts of the C-methylated isoflavone compounds are selected from organic or inorganic acid salts of the C-methylated isoflavone compounds.

4. The application of the C-methylated isoflavone compound according to claim 3 in the preparation of antitumor drugs, characterized in that, The inorganic acid salt of the C-methylated isoflavone compound is selected from the hydrochloride, hydrobromide, or sulfate of the C-methylated isoflavone compound; the organic acid salt of the C-methylated isoflavone compound is selected from the maleate, oxalate, toluenesulfonate, fumarate, or tartrate of the C-methylated isoflavone compound.

5. The use of the C-methylated isoflavone compound according to any one of claims 1-4 in the preparation of antitumor drugs, characterized in that, The antitumor drugs also include pharmaceutically acceptable carriers and / or excipients.

6. The use of the C-methylated isoflavone compound according to any one of claims 1-4 in the preparation of antitumor drugs, characterized in that, The dosage form of the antitumor drug is selected from capsules, granules, injections, tablets, aerosols, oral liquids, or sustained-release formulations.

7. The use of the C-methylated isoflavone compound according to any one of claims 1-4 in the preparation of antitumor drugs, characterized in that, The C-methylated isoflavone compounds were extracted from Polygonatum odoratum.

8. The use of the C-methylated isoflavone compound according to any one of claims 1-4 in the preparation of antitumor drugs, characterized in that, The extraction method for the C-methylated isoflavone compounds is as follows: A. After drying and pulverizing the rhizomes of Polygonatum multiflorum, extract them with an alcohol solvent aqueous solution. Concentrate the extract to remove the solvent and obtain the concentrate. B. Add water to the concentrate for mixing and dispersion, extract with petroleum ether, then extract with ethyl acetate, collect the ethyl acetate phase, elute by column chromatography on a silica gel column, collect the eluent containing compounds of formula 1 and formula 2, and then purify and separate to obtain compounds of formula 1 and formula 2 of C-methylated isoflavone compounds.

9. The application of the C-methylated isoflavone compound according to claim 8 in the preparation of antitumor drugs, characterized in that, The alcohol solvent mentioned in step A is selected from methanol, ethanol or propanol.

10. The application of the C-methylated isoflavone compound according to claim 8 in the preparation of antitumor drugs, characterized in that, The column chromatography described in step B uses a gradient elution with dichloromethane-ethyl acetate eluent.

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