A dihydroisoflavone compound, a preparation method and application thereof, a derivative of the dihydroisoflavone compound and application thereof, and a pharmaceutical composition

Abstract

The application provides a dihydroisoflavone compound and a preparation method and application thereof, a derivative of the dihydroisoflavone compound and application thereof, and a pharmaceutical composition, relates to the technical field of medicines.The dihydroisoflavone compound and the derivative thereof provided by the application have a significant inhibitory effect on triple-negative breast cancer cells, the half-inhibitory concentration value of the dihydroisoflavone compound on the triple-negative breast cancer cells is 0.86 muM, which is equivalent to that of a positive drug, i.e., paclitaxel, thereby providing a new selection, a new idea and technical support for developing a therapeutic drug for triple-negative breast cancer, and having important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to a dihydroisoflavone compound and its preparation method and application, derivatives of the dihydroisoflavone compound and their application, and pharmaceutical compositions. Background Technology

[0002] Malignant tumors are common and prevalent diseases that seriously threaten human health and life. Breast cancer, as the malignant tumor with the highest incidence and mortality rate among women worldwide, has always received widespread attention. Triple-negative breast cancer is a special type of breast carcinoid, accounting for approximately 15-20% of all breast cancers. Triple-negative breast cancer is more aggressive, develops rapidly, has a high recurrence rate, and a poor prognosis, making it the most malignant type of breast cancer. Compared with other breast cancer subtypes, triple-negative breast cancer lacks immunohistochemical expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2, resulting in a lack of suitable therapeutic targets. Commonly used endocrine therapy and targeted therapy have relatively poor effects; for example, tumor inhibitors acting on the PSRP and PI3K / AKT pathways have unsatisfactory prognostic effects, and there is a lack of effective treatment methods. Currently, chemotherapy remains the main method of systemic treatment and an important cornerstone of the systemic treatment regimen for triple-negative breast cancer. Therefore, there is an urgent need to research and develop drugs that can effectively treat triple-negative breast cancer.

[0003] Corethrodendron scoparium, a leguminous plant, is an excellent sand-fixing plant. Its young branches and leaves are high-quality fodder, its wood is durable and long-lasting fuel, its flowers are an excellent source of nectar, and its seeds are high-quality concentrated feed and oilseeds. Currently, there are no published studies on dihydroisoflavone compounds extracted from Corethrodendron scoparium that have therapeutic effects on triple-negative breast cancer. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a dihydroisoflavone compound, its preparation method and application, derivatives of the dihydroisoflavone compound and their applications, and pharmaceutical compositions. The dihydroisoflavone compound provided by this invention has a significant inhibitory effect on triple-negative breast cancer cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a dihydroisoflavonoid compound having the structure shown in Formula I:

[0007]

[0008] This invention also provides a method for preparing the dihydroisoflavone compounds described in the above technical solution, comprising the following steps:

[0009] (1) Extracting Astragalus membranaceus from dwarf rock using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80-100%;

[0010] (2) The alcohol extract is extracted using a mixed extractant of organic extractant and water, and the resulting organic extract phase is concentrated to obtain an organic extract phase extract; the organic extractant includes ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixed extractant is 1:0.8 to 1.2.

[0011] (3) The organic extract is subjected to a first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is a first gradient elution; the eluent used in the first gradient elution includes a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2 to 50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0 to 0:100;

[0012] (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.1; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution included dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0 to 0:100;

[0013] (5) The component Fr.8.1 was subjected to gel column chromatography to obtain component Fr.8.1.2; the elution method of the gel column chromatography was isocratic elution, and the eluent used for the isocratic elution included a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 1:0.5 to 1.5;

[0014] (6) The component Fr.8.1.2 is separated by a reverse-phase column to obtain the dihydroisoflavones; the eluent used for the reverse-phase column separation includes an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol is 85-100%.

[0015] Preferably, the alcohol extraction includes soaking extraction; the temperature of the alcohol extraction is 15-40°C, the number of alcohol extractions is 2-4 times, and the time for each alcohol extraction is 1-5 days;

[0016] The dry weight of the nephrite astragalus and the volume ratio of the first low-grade alcohol aqueous solution used for a single alcohol extraction are 1 kg: 2-20 L.

[0017] Preferably, the lower alcohols in the first and second aqueous solutions of lower alcohols independently include methanol and / or ethanol.

[0018] Preferably, during the first gradient elution process, the volume ratio of petroleum ether to ethyl acetate is 98:2, 95:5, 90:0, 80:20, 70:30, 60:40 and 50:50, respectively, and the volume ratio of dichloromethane to methanol is 100:0, 90:10, 80:20 and 0:100, respectively.

[0019] Preferably, the volume ratio of dichloromethane to methanol during the second gradient elution process is 100:0, 70:30, 50:50, 30:70, and 0:100, respectively.

[0020] The present invention also provides derivatives of the dihydroisoflavone compounds described in the above technical solutions, characterized in that the derivatives include pharmaceutically acceptable salts.

[0021] The present invention also provides the application of the dihydroisoflavone compounds described in the above technical solution or derivatives of the dihydroisoflavone compounds described in claim 7 in the preparation of antitumor drugs.

[0022] Preferably, the tumor includes triple-negative breast cancer.

[0023] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the dihydroisoflavone compounds described in the above-described technical solutions and / or derivatives of the dihydroisoflavone compounds described in the above-described technical solutions.

[0024] The dihydroisoflavone compounds and their derivatives with the structure shown in Formula I provided by this invention have a significant inhibitory effect on triple-negative breast cancer cells, providing new options, new ideas, and technical support for the development of therapeutic drugs for triple-negative breast cancer, and have important clinical application value. As shown in the test results of the examples, the half-maximal inhibitory concentration (WMC) of the dihydroisoflavone compounds provided by this invention against triple-negative breast cancer cells MDA-MB-231 is 0.86 μM, which is comparable to that of the positive control drug paclitaxel.

[0025] This invention provides a method for preparing dihydroisoflavone compounds using Astragalus membranaceus as the raw material, through alcohol extraction, extraction, silica gel column chromatography, and reversed-phase chromatography. The preparation method provided by this invention is simple, easy to operate, has low production costs, is environmentally friendly, and suitable for industrial production. Attached Figure Description

[0026] Figure 1 The ultraviolet spectrum of the isoflavone compound (GCS103) prepared in Example 1;

[0027] Figure 2 The infrared spectrum of GCS103 prepared in Example 1;

[0028] Figure 3 The nuclear magnetic resonance H-spectrum (500MHz, CDCl3) of GCS103 prepared in Example 1;

[0029] Figure 4 Nuclear magnetic resonance C-spectrum and DEPT spectrum (125MHz, CDCl3) of GCS103 prepared in Example 1;

[0030] Figure 5 HMBC spectrum (500 / 125MHz, CDCl3) of GCS103 prepared in Example 1;

[0031] Figure 6 The HRESIMS spectrum of GCS103 prepared in Example 1. Detailed Implementation

[0032] This invention provides a dihydroisoflavonoid compound having the structure shown in Formula I:

[0033]

[0034] The dihydroisoflavone compounds and their derivatives with the structure shown in Formula I provided by this invention have a significant inhibitory effect on triple-negative breast cancer cells, providing new options, new ideas and technical support for the development of therapeutic drugs for triple-negative breast cancer, and have important clinical application value.

[0035] This invention also provides a method for preparing the dihydroisoflavone compounds described in the above technical solution, comprising the following steps:

[0036] (1) Extracting Astragalus membranaceus from dwarf rock using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80-100%;

[0037] (2) The alcohol extract is extracted using a mixed extractant of organic extractant and water, and the resulting organic extract phase is concentrated to obtain an organic extract phase extract; the organic extractant includes ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixed extractant is 1:0.8 to 1.2.

[0038] (3) The organic extract is subjected to a first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is a first gradient elution; the eluent used in the first gradient elution includes a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2 to 50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0 to 0:100;

[0039] (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.1; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution included dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0 to 0:100;

[0040] (5) The component Fr.8.1 was subjected to gel column chromatography to obtain component Fr.8.1.2; the elution method of the gel column chromatography was isocratic elution, and the eluent used for the isocratic elution included a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 1:0.5 to 1.5;

[0041] (6) The component Fr.8.1.2 is separated by a reverse-phase column to obtain the dihydroisoflavones; the eluent used for the reverse-phase column separation includes an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol is 85-100%.

[0042] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0043] This invention utilizes a first low-grade alcohol aqueous solution to extract Astragalus membranaceus from dwarf rock to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80-100%.

[0044] In this invention, the *Astragalus membranaceus* twig is preferably the rhizome of *Astragalus membranaceus* twig, which is preferably dried and cut into segments before use to obtain *Astragalus membranaceus* segments. In this invention, the drying temperature is preferably 25–37°C, and in specific embodiments, it can be 25°C, 28°C, 30°C, 32°C, 35°C, or 37°C. This invention does not have a specific limitation on the drying time; drying to a constant weight is sufficient. In this invention, the length of the *Astragalus membranaceus* segments is preferably 1–2 cm, and in specific embodiments, it can be 1 cm, 1.5 cm, or 2 cm.

[0045] In this invention, the volume fraction of the lower alcohol in the first aqueous solution of lower alcohol is 80-100%, and in specific embodiments it can be 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98% or 100%; the lower alcohol preferably includes methanol and / or ethanol.

[0046] In this invention, the preferred ratio of the dry weight of the nephrite astragalus to the volume of the first low-grade alcohol aqueous solution used for a single alcohol extraction is 1 kg: 2 to 20 L. In specific embodiments, it can be 1 kg: 2 L, 1 kg: 5 L, 1 kg: 8 L, 1 kg: 10 L, 1 kg: 12 L, 1 kg: 15 L, 1 kg: 18 L, or 1 kg: 20 L.

[0047] In this invention, the alcohol extraction preferably includes soaking extraction; the temperature of the alcohol extraction is preferably 15-40°C, and in specific embodiments it can be 15°C, 20°C, 25°C, 30°C, 35°C or 40°C; the number of alcohol extractions is preferably 2-4 times, more preferably 3 times; the time for a single alcohol extraction is preferably 1-5 days, and in specific embodiments it can be 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days or 5 days.

[0048] Following the alcohol extraction, the present invention preferably further includes: performing solid-liquid separation on the alcohol extraction system obtained from the alcohol extraction, and concentrating the resulting alcohol extract to obtain the alcohol extract. The present invention does not have specific limitations on the concentration; any concentration method well-known to those skilled in the art that can remove the solvent can be used, such as vacuum concentration.

[0049] After obtaining the alcohol extract, the present invention uses a mixed extractant of organic extractant and water to extract the alcohol extract, and concentrates the obtained organic extract phase to obtain an organic extract extract paste. In the present invention, the organic extractant preferably includes ethyl acetate and / or petroleum ether, more preferably ethyl acetate. In the present invention, the volume ratio of organic extractant to water in the mixed extractant is preferably 1:0.8 to 1.2, and in specific embodiments it can be 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2. The present invention does not have a special limitation on the concentration, as long as a concentration method well known to those skilled in the art can be used to remove the solvent, such as vacuum concentration.

[0050] After obtaining the organic extract phase extract, the present invention performs a first silica gel column chromatography on the organic extract phase extract to obtain component Fr.8. In the present invention, the elution method of the first silica gel column chromatography is a first gradient elution; the eluent used in the first gradient elution includes, in sequence, a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2 to 50:50, and in specific embodiments, the volume ratio can be 98:2, 95:5, 90:0, 80:20, 70:30, 60:40, and 50:50; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0 to 0:100, and in specific embodiments, the volume ratio can be 100:0, 90:10, 80:20, and 0:100; the particle size of the silica gel used in the first silica gel column chromatography is preferably 200 to 300 mesh. In this invention, 11 components are obtained during the first silica gel column chromatography process, which are sequentially named component Fr.1, component Fr.2, component Fr.3, component Fr.4, component Fr.5, component Fr.6, component Fr.7, component Fr.8, component Fr.9, component Fr.10 and component Fr.11.

[0051] After obtaining component Fr.8, the present invention performs a second silica gel column chromatography to obtain component Fr.8.1. In the present invention, the elution method of the second silica gel column chromatography is a second gradient elution, and the eluent used in the second gradient elution includes dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 100:0 to 0:100. In specific embodiments, the volume ratio can be 100:0, 70:30, 50:50, 30:70, and 0:100, respectively. The preferred particle size of the silica gel used in the second silica gel column chromatography is 200-300 mesh. In the present invention, six components are obtained during the second silica gel column chromatography, which are sequentially designated as component Fr.8.1, component Fr.8.2, component Fr.8.3, component Fr.8.4, component Fr.8.5, and component Fr.8.6.

[0052] After obtaining component Fr.8.1, the present invention performs gel column chromatography on component Fr.8.1 to obtain component Fr.8.1.2. In the present invention, the elution method of the gel column chromatography is isocratic elution, and the eluent used for isocratic elution includes a dichloromethane-methanol mixed solution. The volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 1:0.5 to 1.5, and in specific embodiments it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5. In the present invention, the gel packing material for the gel column chromatography preferably includes Sephadex LH-20. In this invention, the gel column chromatography yields four components, which are denoted as component Fr.8.1.1, component Fr.8.1.2, component Fr.8.1.3 and component Fr.8.1.4, respectively.

[0053] After obtaining component Fr.8.1.2, the present invention performs reversed-phase chromatography column separation on component Fr.8.1.2 to obtain the dihydroisoflavone compound. In the present invention, the eluent used for the reversed-phase chromatography column separation comprises an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 85%–100%, and in specific embodiments it can be 85%, 88%, 90%, 92%, 95%, 98%, or 100%; the lower alcohol preferably includes methanol and / or ethanol. In the present invention, the chromatographic column used for the reversed-phase chromatography column separation preferably includes an RP18 column or a 5C column. 18 -AR-II chromatographic column.

[0054] This invention uses Astragalus membranaceus dentata as the raw material, and dihydroisoflavone compounds can be prepared by alcohol extraction, extraction, silica gel column chromatography, and reversed-phase chromatography. The preparation method provided by this invention is simple, easy to operate, low in production cost, environmentally friendly, and suitable for industrial production.

[0055] This invention also provides derivatives of the dihydroisoflavone compounds described in the above-described technical solutions, wherein the derivatives comprise pharmaceutically acceptable salts. In this invention, the pharmaceutically acceptable salts preferably comprise organic acid salts or metal salts; the organic acid salts preferably comprise acetate, citrate, or oxalate; the metal salts preferably comprise sodium or potassium salts. In this invention, the salt-forming site of the dihydroisoflavone compounds preferably comprises a phenolic hydroxyl group.

[0056] In this invention, the metal salt is preferably obtained by reacting a dihydroisoflavone compound with a base to form a salt; the base preferably includes NaOH or KOH. In this invention, the organic acid salt is preferably obtained by reacting the metal salt with an organic acid through ion exchange or metathesis reaction; the organic acid preferably includes acetic acid, citric acid, or oxalic acid.

[0057] This invention provides the application of the dihydroisoflavone compounds or derivatives thereof described in the above-mentioned technical solutions in the preparation of antitumor drugs. In this invention, the tumor preferably includes triple-negative breast cancer; the triple-negative breast cancer cells preferably include MDA-MB-231.

[0058] The dihydroisoflavone compounds and their derivatives with the structure shown in Formula I provided by this invention have a significant inhibitory effect on triple-negative breast cancer cells, providing new options, new ideas and technical support for the development of therapeutic drugs for triple-negative breast cancer, and have important clinical application value.

[0059] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the dihydroisoflavone compounds described in the above-described technical solutions and / or derivatives of the dihydroisoflavone compounds described in the above-described technical solutions.

[0060] The present invention does not have any particular limitation on the pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0061] In this invention, the mass content of the active component in the pharmaceutical composition is preferably 0.1% to 99%, and in specific embodiments it can be 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0062] In this invention, the dosage form of the pharmaceutical composition preferably includes oral or injectable formulations, and in specific embodiments may be powder, tablet, sugar-coated form, capsule, solution, syrup or drop pill.

[0063] To further illustrate the present invention, the following detailed descriptions, in conjunction with embodiments, describe the dihydroisoflavone compounds, their preparation methods and applications, derivatives of dihydroisoflavone compounds and their applications, and pharmaceutical compositions provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0064] In the following examples, the particle size of the packing material used in the first, second, and third silica gel column chromatography was 200–300 mesh. The reversed-phase column chromatography used was a 5C column. 18 -AR-II chromatographic column (specification: 10mm ID×250mm; manufacturer: COSMOSIL).

[0065] MDA-MB-231 cells were purchased from Wuhan Nopsy Life Science Technology Co., Ltd.

[0066] Example 1

[0067] Preparation of dihydroisoflavone compounds (denoted as GCS103):

[0068] S1: The rhizomes of Astragalus membranaceus were dried to constant weight at 25℃ and then cut into 1cm segments. 20kg of the obtained Astragalus membranaceus segments were soaked in 95v / v% methanol aqueous solution at 30℃ for extraction. The extraction was repeated 3 times. The amount of methanol aqueous solution used in each soaking extraction was 40L and the soaking extraction time was 3 days. The extracts were combined and concentrated under reduced pressure at 40℃ until the solvent was removed to obtain 1.3kg of alcohol extract.

[0069] S2: Extract the alcohol extract with a mixed solvent of ethyl acetate and water (volume ratio 1:1), and concentrate the obtained ethyl acetate phase under reduced pressure at 40°C until the solvent is removed to obtain an ethyl acetate extract.

[0070] S3: The ethyl acetate phase extract was subjected to first silica gel column chromatography. The first silica gel column chromatography was performed by gradient elution with a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution to obtain 11 fractions, denoted as fractions Fr.1 to Fr.11. The 8th fraction (fraction Fr.8) was collected. The specific gradient elution conditions were as follows: the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution was 98:2, 95:5, 90:0, 80:20, 70:30, 60:40 and 50:50, respectively; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 100:0, 90:10, 80:20 and 0:100, respectively.

[0071] S4: Perform a second silica gel column chromatography on fraction Fr.8; the second silica gel column chromatography uses a dichloromethane-methanol mixed solvent as the eluent for gradient elution to obtain 6 fractions (denoted as fractions Fr.8.1 to Fr.8.6), and collect the first fraction (fraction Fr.8.1); the volume ratio of dichloromethane to methanol during the gradient elution process is 100:0, 70:30, 50:50, 30:70 and 0:100 respectively.

[0072] S5: Using a 1:1 volume ratio dichloromethane-methanol mixed solvent as the eluent, fraction Fr.8.1 was subjected to Sephadex LH-20 gel column chromatography to obtain 4 fractions (denoted as fractions Fr.8.1.1 to Fr.8.1.4), and the second fraction (fraction Fr.8.1.2) was collected;

[0073] S6: The fraction Fr.8.1.2 was separated by reversed-phase chromatography using 85 v / v% methanol-water as eluent to obtain dihydroisoflavones (1.8 mg).

[0074] Results analysis:

[0075] The GCS103 prepared in Example 1 was subjected to mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, and NMR analysis. The results are shown in Table 1 and 2. Figures 1-6 ,in, Figure 1 The image shows the ultraviolet spectrum of GCS103. Figure 2 The infrared spectrum of GCS103 is shown below. Figure 3 The nuclear magnetic resonance H-spectrum of GCS103 (500MHz, CDCl3) Figure 4 The nuclear magnetic resonance C-spectrum and DEPT spectrum of GCS103 (125MHz, CDCl3, from top to bottom: DEPT-90° spectrum, DEPT-135° spectrum, and C-spectrum) are shown. Figure 5 The HMBC spectrum of GCS103 (500 / 125MHz, CDCl3) Figure 6 The HRESIMS spectrum of GCS103 is shown below. The structural characterization results of GCS103 are as follows: Molecular formula: C 26 H 30 O6; ESIMS(pos.):m / z 461[M+Na] + ;HRESIMS(pos.):m / z 461.1938[M+Na] + (461.1935 calcd for C) 26 H 30 O6Na); UV(MeOH)λ max :203,224,295,341nm; IR(KBr)ν max :3435,2925,1627,1521,1443,1381,1287,1164,1122,1104cm -1 Based on the results of mass spectrometry, ultraviolet, infrared, and nuclear magnetic resonance analysis, the compound GCS103 prepared in Example 1 has the following structure:

[0076]

[0077] Table 1. Nuclear magnetic resonance spectroscopy data (CDCl3)

[0078]

[0079]

[0080] * indicates that chemical shift assignments are interchangeable.

[0081] Test Example 1

[0082] Viability assays of triple-negative breast cancer cells (MDA-MB-231) and human embryonic kidney cells 293 (HEK-293T):

[0083] Triple-negative breast cancer MDA-MB-231 / HEK-293T cells were placed in 96-well plates at a density of 8000 cells per well and incubated at 37°C for 24 h. Cells were then exposed to different concentrations of test compounds for 48 h each. After incubation, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 2 h. Absorbance was then measured at 450 nm using a microplate reader (Molecular Devices, USA). Relative cell viability was calculated, and IC50 was calculated using GraphPadPrism 9.0.0. 50 The values ​​are listed in Table 2. Paclitaxel was used as a positive control.

[0084] Table 2. Cell viability assays for MDA-MB-231 and HEK-293T cells.

[0085]

[0086] SI (Selectivity Index) = IC 50 (Normal cells) / IC 50 (cancer cell)

[0087] As shown in Table 2, compound GCS-103 has significant inhibitory activity against triple-negative breast cancer MDA-MB-231 cells, comparable to that of the positive drug paclitaxel; moreover, it has low cytotoxicity against normal cells HEK-293T, with a selectivity index of 10.67 and a safety margin higher than that of paclitaxel.

[0088] Example 2

[0089] The GCS-103 prepared in Example 1 was used to prepare a pharmaceutical composition.

[0090] (1) Tablets

[0091] Single tablet formulation: compound GCS-103 10mg, lactose 180mg, starch 55mg, magnesium stearate 5mg;

[0092] Preparation method: GCS-103, lactose and starch are mixed and moistened with water. The moistened mixture is sieved and dried. Magnesium stearate is added after sieving. The mixture is then compressed into tablets, each weighing 250 mg, of which 10 mg is GCS-103.

[0093] (2) Capsules

[0094] Formula: Compound GCS-103 100mg, starch 100mg, magnesium stearate capsule shell;

[0095] Preparation method: GCS-103 compound is mixed with starch, sieved, and the resulting mixture is filled into hard gelatin capsules.

[0096] (3) Ampoules

[0097] Formula: Compound GCS-10 32mg, sodium chloride 10mg, water for injection 100mL;

[0098] Preparation method: Dissolve compound GCS-103 and sodium chloride in 100 mL of water for injection, filter, and fill the resulting mixed solution into ampoules under sterile conditions and seal.

[0099] In summary, the dihydroisoflavone compound GCS103 provided by this invention has significant inhibitory activity against triple-negative breast cancer MDA-MB-231 cells, and has potential application value for further development into an anticancer drug.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a dihydroisoflavone compound, characterized in that, Includes the following steps: (1) Extracting Astragalus membranaceus from dwarf spp. using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 98-100%; (2) The alcohol extract is extracted using a mixture of organic extractant and water, and the resulting organic extract is concentrated to obtain an organic extract extract paste; the organic extractant is selected from ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixture is 1:0.8~1.2; (3) The organic extract is subjected to first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is first gradient elution; the eluent used in the first gradient elution is selected from petroleum ether-ethyl acetate mixed solution and dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2~50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0~0:100; (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.1; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution was selected from dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0~0:100; (5) The component Fr.8.1 was subjected to gel column chromatography to obtain component Fr.8.1.2; the elution method of the gel column chromatography was isocratic elution, and the eluent used for the isocratic elution was selected from a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 1:0.5~1.5; (6) The component Fr.8.1.2 was separated by reversed-phase chromatography to obtain dihydroisoflavones; the eluent used for the reversed-phase chromatography was selected from an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol was 85-98%; The lower alcohols in the first and second aqueous solutions of lower alcohols are independently selected from methanol and / or ethanol; The dihydroisoflavone compounds have the structure shown in Formula I: Equation I.

2. The preparation method according to claim 1, characterized in that, The alcohol extraction is a soaking extraction; the temperature of the alcohol extraction is 15~40℃, the number of alcohol extractions is 2~4 times, and the time for a single alcohol extraction is 1~5 days; The dry weight of the nephrite astragalus and the volume ratio of the first low-grade alcohol aqueous solution used for a single alcohol extraction are 1 kg: 2~20 L.

3. The preparation method according to claim 1, characterized in that, During the first gradient elution process, the volume ratios of dichloromethane and methanol were 100:0, 90:10, 80:20, and 0:100, respectively.

4. The preparation method according to claim 1, characterized in that, During the second gradient elution process, the volume ratios of dichloromethane and methanol were 100:0, 70:30, 50:50, 30:70, and 0:100, respectively.

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