Preparation and application of an aromatic butyl ketone compound

Abstract

This invention discloses the preparation and application of an aromatic butyl ketone compound, belonging to the field of pharmaceutical preparation technology. This invention provides a novel aromatic butyl ketone compound obtained from the fermentation extract of the endophytic fungus *Aspergillus niger* YUD22001. Screening experiments on the antitumor cytotoxic activity of this compound showed that it exhibited significant inhibitory effects on lung cancer A-549 cell line, liver cancer SMMC-7721 cell line, breast cancer DMA-MB-231 cell line, and colon cancer SW-480 cell line. The aromatic butyl ketone compound provided by this invention can be used to prepare antitumor drugs, providing a new option for the development of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the preparation and application of aromatic butyl ketone compounds, belonging to the field of pharmaceutical preparation technology. Background Technology

[0002] Plant endophytic fungi are a class of microorganisms that complete their life cycle within plants. Studies have shown that they have various physiological and ecological interactions with their hosts and the environment, resulting in secondary metabolites with novel structures and rich activities, which is of great significance for the development of new drug lead compounds. Through a systematic study of the secondary metabolites of the endophytic fungus *Aspergillus niger* YUD22001, a novel aromatic butyl ketone compound was discovered for the first time. This compound exhibits strong antitumor cytotoxic activity, providing a new lead compound for further development of antitumor drugs and playing an important role in addressing the current situation of limited types and poor efficacy of clinical anticancer drugs. Summary of the Invention

[0003] This invention provides an endophytic fungus derived from Panax notoginseng. The preservation information of the biological material sample involved in this invention is as follows: the reference microorganism (strain) is YUD22001, classified as Aspergillus niger, and was deposited by the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 12, 2023, with accession number GDMCC No: 63726. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Those skilled in the art can obtain this strain by purchase.

[0004] The aromatic butyl ketone compounds of the present invention have the structure shown in Formula I:

[0005]

[0006] The preparation method of the aromatic butyl ketone compounds of the present invention includes the following specific preparation steps:

[0007] (1) Activate the endophytic fungus Aspergillus nigerYUD22001 of Panax notoginseng.

[0008] (2) The activated strain in step (1) is cultured for two generations to obtain the strain fermentation product.

[0009] (3) The bacterial ferment obtained in step (2) is mixed with ester and then subjected to ultrasonic extraction. After standing, the upper organic solution is taken and distilled under reduced pressure until dry to obtain crude extract.

[0010] (4) The crude extract obtained in step (3) is eluted by silica gel column chromatography to obtain eluent 1.

[0011] (5) Elute the eluent 1 obtained in step (4) onto a reverse-phase silica RP-C18 column to obtain eluent 2.

[0012] (6) The eluent 2 obtained in step (5) was purified by dextran gel column and dried to obtain aromatic butyl ketone compounds.

[0013] Preferably, the culture medium used for activating the endophytic fungus Aspergillus nigerYUD22001 in step (1) is potato dextrose agar medium, and the culture conditions are to place it in an incubator at 28℃ for 2 to 3 days.

[0014] Preferably, the endophytic fungus Aspergillus nigerYUD22001 in step (1) was deposited by the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 12, 2023, with accession number GDMCC No: 63726.

[0015] Preferably, in step (2), the culture medium used for both generations of culture is potato dextrose medium, and the inoculation amount is 5% to 10% of the culture medium used. The first generation culture uses activated bacteria, inoculated at an inoculation amount of 5% to 10%, and cultured in a shaker at 200 rpm and 28°C for 2 to 3 days to form the first generation seed culture solution. The second generation culture uses the first generation seed culture solution, inoculated at an inoculation amount of 5% to 10%, and cultured in a shaker at 150 to 250 rpm and 25 to 29°C for 7 to 14 days to obtain Aspergillus ferment.

[0016] Preferably, in step (3), the volume ratio of the fermented product of the strain to the volume of the ester (the ester used here can be liquid, and there are no other requirements) is 1:1 to 1:1.5, the ultrasonic power is 200 to 400W, the ultrasonic time is 10 to 20 minutes, the ultrasonication is performed 3 times, and the next ultrasonication is performed after an interval of 10 to 20 minutes after each ultrasonication is completed; the standing time is 12 hours; the pressure of vacuum distillation is 6 to 12 kPa, and the temperature is 45 to 55℃.

[0017] Preferably, the silica gel column chromatography elution in step (4) involves dissolving the crude extract in methanol solution at a ratio of 200 g / L to obtain a crude extract solution. The crude extract solution is then thoroughly mixed with 200-mesh silica gel, wherein the mass of the silica gel powder is 0.8 to 1.4 times the dry weight of the crude extract. The solvent is removed by vacuum distillation at a pressure of 6 to 12 kPa and a temperature of 45 to 55°C. The column is then packed, and gradient elution is performed sequentially using dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1, respectively. The eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 is selected as eluent 1.

[0018] Preferably, in step (5), the solvent in the washing solution 1 of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 obtained in step (4) is removed by vacuum distillation at a pressure of 6-12 kPa and a temperature of 45-55°C. Methanol solution is added to dissolve the solute (the amount of methanol is not required, as long as the solute is completely dissolved) to obtain a solution. The solution is added to degreased cotton, allowed to stand and evaporate, and then packed into an RP-C18 column. Gradient elution is performed sequentially using methanol-water mixed solutions with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 to obtain methanol-water eluents with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0. The eluent with a volume ratio of 1:1 is selected as the eluent 2.

[0019] Preferably, in step (6), the solvent in the eluent 2 obtained in step (5) with a methanol to water volume ratio of 1:1 is removed by vacuum distillation at a pressure of 6-12 kPa and a temperature of 45-55°C. The solvent is dissolved in methanol solution, purified by dextran gel column at a flow rate of 0.3-0.6 mL / min, and dried to obtain aromatic butyl ketone compounds.

[0020] Preferably, the dextran gel column used in this invention is Sephadex LH-20.

[0021] The application of the aromatic butyl ketone compounds described in this invention in anti-tumor activity shows that these compounds exhibit strong inhibitory activity against lung cancer A-549 cell line, liver cancer SMMC-7721 cell line, breast cancer MDA-MB-231 cell line, and colon cancer SW-480 cell line.

[0022] Beneficial effects of the present invention

[0023] (1) This invention provides an aromatic butanone compound, which is an aryl-substituted 2-butanone compound with good antitumor cytotoxic activity. It can enrich the diversity of aromatic butanone compounds and can also be used to prepare antitumor drugs, providing a new approach for the development and research of antitumor drugs.

[0024] (2) Screening tests on the antitumor cytotoxic activity of aromatic methyl ethyl ketone compounds of the present invention showed that the aromatic methyl ethyl ketone compounds provided by the present invention all exhibited significant inhibitory activity against liver cancer A-549 cell line, lung cancer SMMC-7721 cell line, breast cancer MDA-MB-231 cell line and colon cancer SW-480 cell line.

[0025] (3) The present invention uses bacterial fermentation to prepare aromatic butanone compounds. The preparation method has the advantages of high efficiency, low cost and mild culture conditions, which can be easily industrialized and can also provide a new option for the mass production of aromatic butanone compounds. Attached Figure Description

[0026] Figure 1 The aromatic butyl ketone compounds prepared in Example 1 1 H-NMR spectrum.

[0027] Figure 2 The aromatic butyl ketone compounds prepared in Example 1 13 C-NMR spectrum.

[0028] Figure 3 The aromatic butyl ketone compounds prepared in Example 1 1 H- 1 H COSY spectrum.

[0029] Figure 4 The HMBC spectrum of the syringone compounds prepared in Example 1 is shown.

[0030] Figure 5 The HSQC spectrum of the aromatic butanone compounds prepared in Example 1 is shown.

[0031] Figure 6 The HR-ESI-MS spectrum of the aromatic butanone compounds prepared in Example 1 is shown. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, all reagents used in the present invention are conventional commercially available reagents. The Aspergillus niger YUD22001 strain used in the present invention has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with the accession number GDMCCNo: 63726. Therefore, this strain can be purchased from the Guangdong Provincial Center for Microbial Culture Collection.

[0033] Preparation of culture medium used in the experiment

[0034] Potato glucose medium (PDB medium): Take 200g of washed and peeled potatoes, cut them into pieces about 1cm in diameter, add water and boil until the potato pieces can be pierced with a glass rod. Filter the filtrate with gauze, add 20g of glucose to the filtrate, add water to 1000mL, place the prepared culture in a high-temperature sterilizer at 121℃ for 30 minutes, remove it, cool it and store it for later use.

[0035] Potato glucose agar medium (PDA medium) is available for purchase.

[0036] In this invention, unless otherwise specified, all required materials or reagents are commercially available products well known to those skilled in the art.

[0037] Example 1

[0038] The specific steps for preparing an aromatic butanone compound are as follows:

[0039] (1) Inoculate the endophytic fungus Aspergillus nigerYUD22001 of Panax notoginseng onto PDA medium and incubate it in an incubator for 2 days at a temperature of 28℃.

[0040] (2) The activated endophytic fungus Aspergillus nigerYUD22001 from step (1) was inoculated into PDB medium at a rate of 10% and cultured at a temperature of 28°C and a rotation speed of 200 rpm for 3 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation PDB medium at a rate of 10% and cultured at a temperature of 28°C and a rotation speed of 200 rpm for 7 days to obtain the strain ferment.

[0041] (3) The strain fermentation product obtained in step (2) is thoroughly mixed with ethyl acetate. The mixing ratio is 1:1 (volume ratio of Aspergillus fermentation product to ethyl acetate). The mixed solution is placed in an ultrasonic extractor for ultrasonic extraction. The ultrasonic extraction conditions are 300W power for 15 minutes, a total of 3 times, with an interval of 10 minutes between each time. After ultrasonic extraction, the mixture is allowed to stand for 12 hours. The upper organic phase solution is taken and subjected to vacuum distillation until dry. The pressure of vacuum distillation is 9 kPa and the temperature is 50℃ to obtain the crude extract.

[0042] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (200 mesh). The mass of the silica gel used is 1.2 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 9 kPa and a temperature of 50°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 as eluent 1.

[0043] (5) Remove the solvent from the eluent 1 obtained in step (4) by vacuum distillation at a pressure of 9 kPa and a temperature of 50 °C. Dissolve the solute in methanol solution to obtain a solution. Add the solution dropwise onto the defatted cotton in three portions, let it stand to evaporate, and then pack it into a reversed-phase silica RP-C18 column. Perform gradient elution with methanol-water mixed solutions with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in sequence to obtain methanol-water eluents with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in the same volume. Select the eluent with a volume ratio of 1:1 in the methanol-water mixture as eluent 2.

[0044] (6) The solvent of the eluent 2 obtained in step (5) was removed by vacuum distillation at a pressure of 9 kPa and a temperature of 50 °C. The solvent was methanol, and the solution was purified using a dextran gel (Sephadex LH-20) column at a flow rate of 0.4 mL / min. The solution was dried to obtain aromatic butyl ketone compounds.

[0045] The aromatic methyl ethyl ketone compounds obtained in this embodiment were structurally identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). The results are as follows: Figures 1-6 As shown.

[0046] From HSQC spectrum and 13The chemical shifts δ of H and C of this aromatic cyclobutanone compound can be obtained from the C-spectrum, as shown in Table 1.

[0047] Table 1. Aromatic butyl ketone compounds prepared in Example 1 13 C and 1 HNMR data, DMSO as solvent

[0048]

[0049]

[0050] from Figures 1-6 As can be seen from the data, the HR-ESI-MS m / z of aromatic butyl ketone compounds is: [M+H] + The quasi-molecular ion peak is 209.1171 ( Figure 6 (C) 12 H 17 O3[M+H] + Calculated value: 209.1179), indicating that its molecular formula is C. 12 H 16 O3 contains five degrees of unsaturation. Through the analysis of compounds... 1 H( Figure 1 ), 13 C( Figure 2 This indicates that the compound contains 12 carbon signals and includes one methoxy group (δ). C 55.8, δ H 3.64), two methyl groups (δ) C 16.0, 7.7, δ H 2.10, 0.89), two methylene groups (δ C 43.2, 34.3, δ H 3.50, 2.40), two methines (δ C 117.4, 113.6, δ H 6.53, 6.69), and 5 quaternary carbons (δ C 208.6, 121.4, 148.6, 122.7, 149.7).

[0051] Analysis of the 2D nuclear magnetic resonance spectra of aromatic butyl ketone compounds, as shown in the HMBC diagram, reveals that H-4(δ) H 6.53) to C-6 (δ) C 122.7), C-8 (δ) C The correlation of 149.7), H-7 (δ) H 6.69) to C-10(δ C 16.0), C-8 (δ) CThe correlation with 149.7 indicates that the compound contains a tetrasubstituted aromatic ring unit. H-2(δ) H 3.50) to C-8 (δ) C 149.7), H-9 (δ) H 3.64) to C-8(δ) C The HMBC correlation of 149.7) indicates that the methoxy group is linked to C-8, and H-7 (δ) H 6.69) and C-8(δ) C 149.7), C-10 (δ) C The HMBC correlation of 16.0) indicates that the methyl group is attached to C-6. H-11(δ) H 2.40) to C-1(δ C 208.6), C-12 (δ) C The HMBC correlation in 7.7) indicates that C-11 is connected to C-1 and C-12. This conclusion can also be reached through the correlation between H-11 and H-12. 1 H- 1 H COSY( Figure 3 This is used to prove the point. Based on the molecular formula, the planar structure of the aromatic butanone compounds was thus derived. Therefore, the planar structure of the aromatic butanone compounds provided by this invention is determined to be a 2-butanone compound containing a benzene ring substitution.

[0052] In summary, the structural formula of the compound prepared in Example 1 can be determined as follows:

[0053]

[0054] Example 2

[0055] The specific steps for preparing an aromatic butanone compound are as follows:

[0056] (1) Inoculate the endophytic fungus Aspergillus nigerYUD22001 of Panax notoginseng onto PDA medium and incubate it in an incubator for 3 days at a temperature of 28℃.

[0057] (2) The activated endophytic fungus Aspergillus nigerYUD22001 from step (1) was inoculated into PDB medium at a rate of 5% and cultured at a temperature of 28°C and a rotation speed of 150 rpm for 2 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation PDB medium at a rate of 5% and cultured at a temperature of 25°C and a rotation speed of 150 rpm for 14 days to obtain the strain ferment.

[0058] (3) The strain fermentation product obtained in step (2) is thoroughly mixed with ethyl acetate. The mixing ratio is 1:1.5 (volume ratio of Aspergillus fermentation product to ethyl acetate). The mixed solution is placed in an ultrasonic extractor for ultrasonic extraction. The ultrasonic extraction conditions are 200W power for 20 minutes, a total of 3 times, with an interval of 15 minutes between each time. After ultrasonic extraction, the mixture is allowed to stand for 12 hours. The upper organic phase solution is taken and subjected to vacuum distillation until dry. The pressure of vacuum distillation is 6 kPa and the temperature is 55℃ to obtain crude extract.

[0059] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (200 mesh). The mass of the silica gel used is 0.8 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 6 kPa and a temperature of 55°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 as eluent 1.

[0060] (5) Remove the solvent from the eluent 1 obtained in step (4) by vacuum distillation at a pressure of 6 kPa and a temperature of 55 °C. Dissolve the solute in methanol solution to obtain a solution. Add the solution dropwise onto the defatted cotton in three portions, let it stand to evaporate, and then pack it into a reversed-phase silica RP-C18 column. Perform gradient elution with methanol-water mixed solutions with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in sequence to obtain methanol-water eluents with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in the same volume. Select the eluent with a volume ratio of 1:1 in the methanol-water mixture as eluent 2.

[0061] (6) The solvent of the eluent 2 obtained in step (5) was removed by vacuum distillation at a pressure of 6 kPa and a temperature of 55 °C. The solvent was methanol, and the solution was purified using a dextran gel (Sephadex LH-20) column at a flow rate of 0.6 mL / min. The solution was dried to obtain aromatic butyl ketone compounds.

[0062] The aromatic methyl ethyl ketone compounds obtained in this example were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry), and their structures were the same as those in Example 1.

[0063] Example 3

[0064] The specific steps for preparing an aromatic butanone compound are as follows:

[0065] (1) Inoculate the endophytic fungus Aspergillus nigerYUD22001 of Panax notoginseng onto PDA medium and incubate it in an incubator for 2 days at a temperature of 28℃.

[0066] (2) The activated endophytic fungus Aspergillus nigerYUD22001 from step (1) was inoculated into PDB medium at a rate of 5% and cultured at a temperature of 28°C and a rotation speed of 250 rpm for 2 days to form a first-generation seed culture. The first-generation seed culture was then inoculated into second-generation PDB medium at a rate of 5% and cultured at a temperature of 29°C and a rotation speed of 250 rpm for 8 days to obtain the strain ferment.

[0067] (3) The strain fermentation product obtained in step (2) is thoroughly mixed with ethyl acetate. The mixing ratio is 1:1 (volume ratio of Aspergillus fermentation product to ethyl acetate). The mixed solution is placed in an ultrasonic extractor for ultrasonic extraction. The ultrasonic extraction conditions are 400W power for 10 minutes, a total of 3 times, with an interval of 20 minutes between each time. After ultrasonic extraction, the mixture is allowed to stand for 12 hours. The upper organic phase solution is taken and subjected to vacuum distillation until dry. The pressure of vacuum distillation is 12 kPa and the temperature is 45℃ to obtain the crude extract.

[0068] (4) Dissolve the crude extract obtained in step (3) in methanol at a concentration of 200 g / L. Mix the dissolved crude extract solution thoroughly with silica gel (200 mesh). The mass of the silica gel used is 1.4 times that of the crude extract. Remove the solvent by vacuum distillation at a pressure of 12 kPa and a temperature of 45°C. Then pack the silica gel column and perform gradient elution with dichloromethane-methanol mixed solutions with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with volume ratios of 1:0, 50:1, 30:1, 10:1, and 0:1. Select the eluent of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 as eluent 1.

[0069] (5) Remove the solvent from the eluent 1 obtained in step (4) by vacuum distillation at a pressure of 12 kPa and a temperature of 45 °C. Dissolve the solute in methanol solution to obtain a solution. Add the solution dropwise onto the defatted cotton in three portions, let it stand to evaporate, and then pack it into a reversed-phase silica RP-C18 column. Perform gradient elution with methanol-water mixed solutions with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in sequence to obtain methanol-water eluents with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0 in the same volume. Select the eluent with a volume ratio of 1:1 in the methanol-water mixture as eluent 2.

[0070] (6) The solvent of the eluent 2 obtained in step (5) was removed by vacuum distillation at a pressure of 12 kPa and a temperature of 45 °C. The solvent was methanol, and the solution was purified by using a dextran gel (Sephadex LH-20) column at a flow rate of 0.3 mL / min. The solution was dried to obtain aromatic methyl ethyl ketone compounds.

[0071] The aromatic methyl ethyl ketone compounds obtained in this example were identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry), and their structures were the same as those in Example 1.

[0072] Example 4

[0073] Antitumor cytotoxic activity assay of aromatic butyl ketone compounds

[0074] (1) Cell seeding: Prepare single cell suspensions of lung cancer A-549 cells, liver cancer SMMC-7721 cells, breast cancer MDA-MB-231 cells, and colon cancer SW-480 cells in culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum. Seed 3,000 to 5,000 cells per well into 96-well plates with a volume of 100 μL per well. The cells should be seeded and cultured 12 to 24 hours in advance.

[0075] (2) Add the aromatic methyl ethyl ketone compound solution prepared in Example 1: The aromatic methyl ethyl ketone compound was dissolved in DMSO, and the compound was screened again at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0.064 μM, with a final volume of 200 μl per well. Each treatment was performed in 3 replicates.

[0076] (3) Color development: After culturing at 37℃ for 48 hours, discard the culture medium in the wells of adherent cells, add 20 μL of MTS solution and 100 μL of culture medium (DMEM or RMPI1640) to each well, set up 3 blank parallels (a mixture of 20 μL of MTS solution and 100 μL of culture medium), and continue incubation for 2 to 4 hours to allow the reaction to proceed fully before measuring the light absorbance.

[0077] (4) Colorimetric analysis: Using a wavelength of 492 nm, the absorbance values ​​of each well were read using a multi-functional microplate reader (MULTISKAN FC). The results were recorded, and after data processing, a cell growth curve was plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compound was calculated using the Reed and Muench method. 50 value.

[0078] (5) Positive control compound: Cisplatin (DDP) was used as the positive control compound in each experiment. Cell growth curves were plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compound was calculated using the Reed and Muench method. 50 value.

[0079] The experimental results are shown in Table 2. As can be seen from Table 2, in the experiment testing the antitumor cytotoxic activity of aromatic methyl ethyl ketone compounds (with cisplatin as the positive control), these compounds exhibited strong inhibitory activity against lung cancer A-549 cells, liver cancer SMMC-7721 cells, breast cancer MDA-MB-231 cells, and colon cancer SW-480 cells. Compared with the positive control DDP, the aromatic methyl ethyl ketone compounds showed comparable or even superior inhibitory activity against these four human tumor cell types. Therefore, the aromatic methyl ethyl ketone compounds provided by this invention demonstrate significant research value in the development of antitumor drugs. Furthermore, the method for obtaining aromatic methyl ethyl ketone compounds with antitumor cytotoxic activity through bacterial fermentation not only achieves high efficiency, low cost, and convenient operation, but also provides an option for further mass production of aromatic methyl ethyl ketone compounds.

[0080] Table 2 IC50 of cytotoxic activity of aromatic methyl ketone compounds 50 Value (μM)

[0081]

[0082] 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 an aromatic butanone compound, characterized in that: The specific preparation steps are as follows: (1) Endophytic fungi of Panax notoginseng Aspergillus niger YUD22001 strain was activated; (2) The activated strain from step (1) is cultured for two generations to obtain the strain fermentation product; (3) The bacterial ferment obtained in step (2) is mixed with ester and then subjected to ultrasonic extraction. After standing, the upper organic solution is taken and subjected to vacuum distillation until the solvent is evaporated to dryness to obtain crude extract. (4) The crude extract obtained in step (3) was eluted by silica gel column chromatography to obtain eluent 1; (5) Elute the eluent 1 obtained in step (4) onto a reverse-phase silica RP-C18 column to obtain eluent 2; (6) The eluent 2 obtained in step (5) is purified by dextran gel column and dried to obtain aromatic butyl ketone compounds, which have the structure shown in Formula I; Equation I; In step (1), the endophytic fungi of Panax notoginseng Aspergillus niger YUD22001 was deposited by the Guangdong Provincial Center for Microbial Culture Collection on August 12, 2023, with accession number GDMCC No: 63726.

2. The method for preparing aromatic butyl ketone compounds according to claim 1, characterized in that: In step (1), the endophytic fungi of Panax notoginseng Aspergillus niger The culture medium used for activating the YUD22001 strain was potato dextrose agar, and the culture conditions were as follows: place it in an incubator at 28 ℃ and culture for 2-3 days.

3. The method for preparing aromatic butylone compounds according to claim 1, characterized in that: In step (2), the culture medium used for both generations of culture is potato dextrose medium. The first generation culture uses activated bacteria, and the inoculum amount is 5%~10% of the culture medium. The culture conditions are to culture in a shaker at 200 rpm and 28℃ for 2~3 days to form the first generation seed culture. The second generation culture uses the first generation seed culture, and the inoculum amount is 10% of the culture medium. The culture conditions are to culture in a shaker at 150~250 rpm and 25~29℃ for 7~14 days to obtain the fermentation product.

4. The method for preparing aromatic butyl ketone compounds according to claim 1, characterized in that: In step (3), the volume ratio of the fermented product to the ester is 1:1 to 1:1.5, the ultrasonic power is 200 to 400W, the ultrasonic time is 10 to 20 minutes, and the ultrasonication is performed 3 times. After each ultrasonication, there is an interval of 10 to 20 minutes before the next ultrasonication is performed. The standing time is 12 hours. The pressure of vacuum distillation is 6 to 12 kPa, and the temperature is 45 to 55℃.

5. The method for preparing aromatic butylone compounds according to claim 1, characterized in that: The specific operation process of silica gel column chromatography elution in step (4) is as follows: the crude extract is fully dissolved in methanol solution at a ratio of 200 g / L to obtain crude extract solution; the crude extract solution is fully mixed with 200 mesh silica gel, wherein the mass of silica gel powder is 0.8 to 1.4 times the dry weight of the crude extract; the solvent is removed by vacuum distillation at a pressure of 6 to 12 kPa and a temperature of 45 to 55 °C; the silica gel column is loaded, and gradient elution is performed sequentially using dichloromethane-methanol mixed solutions with a volume ratio of dichloromethane to methanol of 1:0, 50:1, 30:1, 10:1, and 0:1 to obtain dichloromethane-methanol eluents with a volume ratio of dichloromethane to methanol of 1:0, 50:1, 30:1, 10:1, and 0:1, respectively. The eluent of the dichloromethane-methanol mixed solution with a volume ratio of dichloromethane to methanol of 30:1 is selected as eluent 1.

6. The method for preparing aromatic butylone compounds according to claim 1, characterized in that: In step (5), the solvent in the washing solution 1 of the dichloromethane-methanol mixed solution with a volume ratio of 30:1 obtained in step (4) is removed by vacuum distillation at a pressure of 6~12 kPa and a temperature of 45~55℃. The solute is dissolved in methanol solution to obtain a solution. The solution is added to degreased cotton, allowed to stand and evaporate, and then packed into an RP-C18 column. Gradient elution is performed using methanol-water mixed solutions with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:0, respectively, to obtain methanol-water eluents with volume ratios of 1:4, 2:3, 1:1, 3:2, and 1:

0. The eluent with a volume ratio of 1:1 is selected as eluent 2.

7. The method for preparing aromatic butylone compounds according to claim 1, characterized in that: In step (6), the solvent in the eluent 2 obtained in step (5) with a methanol to water volume ratio of 1:1 is removed by vacuum distillation at a pressure of 6~12 kPa and a temperature of 45~55℃. The solvent is dissolved in methanol solution and purified using a dextran gel column at a flow rate of 0.3~0.6 mL / min. After drying, aromatic butyl ketone compounds are obtained.

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