A benzogchromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, a preparation method thereof, and applications thereof

Abstract

The present invention belongs to the technical field of food chemistry, and particularly relates to a benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and its preparation method and application. The benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity is prepared from the medicinal plant Cassia obtusifolia ( Chamaecrista rotundifolia ) whole plant was used as raw material, and the product was prepared through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation. The product is named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, and has the following structure: #imgabs0#. The antibacterial benzo[g]chromone compound of the present invention has good activity against drug-resistant bacteria and can be used as a lead compound in the development of drugs against drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of phytochemistry, and in particular relates to a benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and a preparation method and application thereof. Background Art

[0002] Cassia tora (scientific name: Chamaecrista rotundifolia Pers. greene is a semi-erect perennial herbaceous plant in the Fabaceae family, Cassia genus. Cassia roundleaf is resistant to acid, drought, and infertility, offers high yields, strong nitrogen fixation, high nutrient content, and is pest and disease-free.

[0003] Years of cultivation have shown that Cassia obtusifolia, as a perennial herb, is suitable for cultivation on mountainous and sloping lands. It helps prevent soil erosion and water loss, and plays a significant role in maintaining ecological balance. Cassia obtusifolia has a crude protein content of approximately 20%, along with high levels of crude fat, nitrogen, phosphorus, and calcium, but a low crude fiber content. Its yield reaches 1,500 kg per mu, equivalent to the crude protein content of 3,000 kg of corn, making it an excellent forage crop with high economic value. Cassia obtusifolia also has extensive medicinal properties, including heat-clearing and detoxifying, diuretic and stranguria-relieving, lipid-lowering, antibacterial, and radiation-resistant properties. However, relatively little research has been conducted on its secondary metabolites. Summary of the Invention

[0004] The first purpose of the present invention is to provide a benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity; the second purpose is to provide a method for preparing the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity; and the third purpose is to provide an application of the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity.

[0005] The first object of the present invention is achieved by: the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity is prepared from the medicinal plant Cassia obtusifolia ( Chamaecrista rotundifolia The whole plant was used as raw material and prepared through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. It is named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, and its English name is: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, with the following structure:

[0006] .

[0007] The second object of the present invention is achieved by using the medicinal plant Cassia obtusifolia ( Chamaecrista rotundifolia ) whole plant is used as raw material, and the preparation is carried out through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, which specifically includes the following steps:

[0008] A. Pretreatment: Grind or cut the whole plant of the raw medicinal plant Cassia tora into segments to obtain material a;

[0009] B. Extraction: Add 2-4 times the mass of organic extraction solvent to material a, soak and extract 3-5 times at room temperature, each extraction time is 24-72 hours, combine the extracts, filter and concentrate to obtain extract b;

[0010] C. Silica gel column chromatography: Add 160-300 mesh silica gel in an amount 2-4 times the weight of extract b to the column and dry-pack it. Use a chloroform-acetone solution with a volume ratio of 1:0-1:2 for gradient elution. Monitor by TLC and combine the same fractions.

[0011] D. High Performance Liquid Chromatography Separation: The eluate obtained by eluting with a 7:3 chloroform-acetone solution was separated and purified by high performance liquid chromatography to obtain the target benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity.

[0012] The specific operations are as follows:

[0013] 1. Extraction: Crush the whole plant of Cassia torata, use high-concentration methanol (w%: 80%-100%), high-concentration ethanol (w%: 80%-100%), or high-concentration acetone (w%: 60%-90%) as the extraction solvent, with the extraction solvent: whole plant of Cassia torata (weight ratio) = 2-4:1, soak for 24 hours to 72 hours, extract 3-5 times, combine the extracts, filter and concentrate to obtain the extract;

[0014] 2. Silica gel column chromatography: Dissolve the extract in 1.5-3 times the weight of pure methanol or pure acetone, then mix with 0.8-1.2 times the weight of 80-100 mesh silica gel. Dry-pack the column with 2-4 times the weight of 160-300 mesh silica gel for silica gel column chromatography. Gradient elution is performed with chloroform-acetone solutions in volume ratios of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2). Combine the same fractions, collect the eluates from each fraction, and concentrate.

[0015] 3. Separation and purification by high performance liquid chromatography: The 7:3 portion of the column chromatography eluate is further separated and purified by high performance liquid chromatography to obtain the benzo[g]chromone compounds.

[0016] HPLC separation and purification was performed using a 21.2 mm × 250 mm, 5 μm C 18 The chromatographic column has a flow rate of 12 mL / min, the mobile phase is 52% methanol, the detection wavelength of the UV detector is 368 nm, 500 μL is injected each time, the chromatographic peak is collected for 30 minutes, and after multiple accumulation, it is evaporated to dryness and then purified by gel to obtain the compound of the present invention.

[0017] The structures of the benzo[g]chromone compounds prepared by the above method were determined by the following method.

[0018] The compound of the present invention is a yellow colloid. 1 H and 13 C NMR spectrum (see Table 1, Figure 1 and Figure 2 ) showed that it contained 16 carbons and 14 hydrogens, including 1 tetrasubstituted naphthalene ring (C-5~C-13, C-4a, C-9a, H-5, H-7, H-8 and H-10), and 1 α,β -unsaturated carbonyl (-OC=CH-CO-, C-2~C-4, H-3), a methyl (C-16 and H3-16), a hydroxymethyl (C-15 and H2-15), and a methoxy ( δ C 56.1 and δ H 3.81 s). Further analysis of its NMR data, α,β The unsaturated carbonyl group is expected to connect to the naphthalene ring to form a pyran ring, forming a benzo[g]chromone skeleton, supporting the presence of ten degrees of unsaturation and two oxidized aromatic carbons (C-2 and C-3). This speculation is further confirmed by HMBC correlations between H-3 and C-2 / C-4 / C-12, H-5 to C-4 / C-6, H-7 to C-13, H-8 and C-14, and H-10 and C-9.

[0019] Table 1. Compounds of the present invention 1 HNMR and 13 C NMR data (CDCl3)

[0020]

[0021] After the parent compound is determined, the remaining substituents (methoxy, methyl and hydroxymethyl) can be regarded as benzo[ g ] substituents on chromone. Methoxy substituted at C-6 position can be replaced by methoxy hydrogen ( δ H3.81 s) and C-6 were confirmed by HMBC correlation. HMBC correlations between H3-16 and C-8 / C-9 / C-14, and between H-8 and C-16, confirmed the methyl substitution at C-9. HMBC correlations between H2-15 and C-2 / C-3, and between H-3 and C-15, confirmed the hydroxymethyl substitution at C-2. Thus, the structure of the compound of the present invention was determined, and it was named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0022] Infrared, UV and mass spectral data of the compound: UV spectrum (methanol), λ max (log ε ) 215 (4.47), 275 (4.02), 368 (3.96) nm; Infrared spectrum (potassium bromide tablet): ν max 3396, 2950, 1662, 1618, 1564, 1457, 1274, 1175, 1043, 938 cm -1 ; 1 H and 13 C NMR data (500 and 125 MHz, (CDCl3), see Table 1; positive ion mode ESIMS m / z 293 [M+Na] + ; Positive ion mode HRESIMS m / z 293.0789 [M+Na] + (Calculated value 293.0784, C 16 H 14 NaO 4） .

[0023] 4. In vitro antibacterial activity detection

[0024] The test strain was a methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from a clinical specimen at Kunming Medical University. The strain was phenotypically oxacillin-resistant (≥4 mg / L) and tested positive for the mecA gene. Antimicrobial susceptibility testing was performed using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). The protocol followed the CLSIM07-A9 standard, and the judgment criteria followed the CLSIM100-S24 standard. The specific method is as follows:

[0025] (1) Preparation of antimicrobial stock solution: The concentration of the antimicrobial stock solution is 2560 μg / mL. Antimicrobial drugs with low solubility may be prepared at a slightly lower concentration. The required amount of antimicrobial solution or powder can be calculated using the formula. The prepared antimicrobial stock solution should be stored below -20°C and should not exceed 6 months.

[0026] (2) Preparation of test bacteria: Use an inoculation loop to pick a single colony from the overnight cultured MH(A) dish and place it in MH(B) medium, calibrate it to a 0.5 McFarland turbidimetric standard, containing approximately 1×108 CFU / ml of bacteria, and then dilute it 100-fold to obtain a culture medium containing approximately 1×10 6 CFU / mL of bacterial solution for future use.

[0027] (3) Dilute the stock solution of antimicrobial drugs (2560 μg / mL) 10-fold to obtain an antimicrobial drug solution with a concentration of 256 μg / mL. Take a sterile 96-well plate, add 200 μL of antimicrobial drugs to the first well, add 100 μL of MH broth culture medium to the second to tenth wells, draw 100 μL from the first well to the second well, mix, draw 100 μL to the third well, and so on, draw 100 μL from the tenth well and discard. At this time, the drug concentrations in each well are: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / mL, add 100 μL of bacterial solution to the eleventh well (positive control), and add 100 μL of MH(B) culture medium to the twelfth well (negative control).

[0028] (4) Add 100 μL of the previously prepared bacterial solution to each well from 1 to 10, so that the final bacterial solution concentration in each tube is approximately 5×10 5 CFU / mL, with drug concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL in wells 1 to 10, respectively. Place the inoculated 96-well plate in a 37°C incubator and observe bacterial growth for 24 hours. Use a standard strain for quality control.

[0029] (5) Result evaluation and interpretation: Before reading and reporting the MIC of the test strain, the growth control tube should be checked for good bacterial growth. The inoculum subculture should also be checked to determine whether it is contaminated and whether the MIC value of the quality control strain is within the quality control range. The tube with the lowest drug concentration that shows no bacterial growth by visual observation is the MIC of the test strain.

[0030] (6) The test results showed that compound (1) has outstanding antibacterial activity and has obvious antibacterial advantages compared with the antibacterial drugs used in clinical treatment of Gram-positive bacteria infections. 90The concentration was 52.84±4.2 μg / mL, with significant antibacterial activity, close to the MIC of levofloxacin. 90 value (>50.84 ± 5.5 μg / mL).

[0031] Compared with the prior art, the present invention has the following outstanding advantages: (1) The compound of the present invention uses the whole plant of Cassia tora as raw material. In order to control soil erosion, Cassia tora is artificially planted on a large scale in barren wastelands and sloping lands, with large biological yield, a very wide source of raw materials, low cost, and easy separation and preparation. (2) The extraction method of the compound of the present invention is simple and easy to separate. (3) The preparation method adopts a combination of conventional column chromatography and high performance liquid chromatography, the compound preparation operation process is simple, the obtained compound of the present invention has high purity, and subsequent industrial production is easy to achieve. (4) The compound of the present invention has low toxicity to animals, is safe to use, and exhibits good activity against drug-resistant bacteria, and can be used as a lead compound for the development of drugs against drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The nuclear magnetic resonance carbon spectrum of the benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of the present invention is as follows: 13 C NMR);

[0033] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of the present invention is ( 1 H NMR);

[0034] Figure 3 Key HMBC correlation diagram of the benzo[g]chromone compounds of the present invention having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity. DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0036] The benzo[g]chromone compounds with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of the present invention are prepared from the medicinal plant Cassia obtusifolia ( Chamaecrista rotundifoliaThe whole plant was used as raw material and prepared through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. It was named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, and its English name is: 2-(hydroxy methyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, with the following structure:

[0037] .

[0038] The preparation method of the benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of the present invention is to prepare the benzo[g]chromone compound with the medicinal plant Cassia obtusifolia ( Chamaecrista rotundifolia ) whole plant is used as raw material, and the preparation is carried out through pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, which specifically includes the following steps:

[0039] A. Pretreatment: Grind or cut the whole plant of the raw medicinal plant Cassia tora into segments to obtain material a;

[0040] B. Extraction: Add 2-4 times the mass of organic extraction solvent to material a, soak and extract 3-5 times at room temperature, each extraction time is 24-72 hours, combine the extracts, filter and concentrate to obtain extract b;

[0041] C. Silica gel column chromatography: Add 160-300 mesh silica gel in an amount 2-4 times the weight of extract b to the column and dry-pack it. Use a chloroform-acetone solution with a volume ratio of 1:0-1:2 for gradient elution. Monitor by TLC and combine the same fractions.

[0042] D. High Performance Liquid Chromatography Separation: The eluate obtained by eluting with a 7:3 chloroform-acetone solution was separated and purified by high performance liquid chromatography to obtain the target benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity.

[0043] The organic extraction solvent in step B is an aqueous methanol solution with a mass concentration of 80% to 100%, an aqueous ethanol solution with a mass concentration of 80% to 100%, or an aqueous acetone solution with a mass concentration of 60% to 90%.

[0044] Step C also includes the step of dissolving the extract b with an organic solvent at a volume of 1.5 to 3 times the weight of the extract b and then adding 80 to 100 mesh silica gel at a volume of 0.8 to 1.2 times the weight of the extract b to mix the sample before loading onto the column.

[0045] The organic solvent is pure methanol, pure ethanol or pure acetone.

[0046] The volume ratios of the chloroform-acetone solution described in step C are 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2.

[0047] The HPLC separation and purification in step D was performed using a 21.2 mm × 250 mm, 5 μm C 18 The chromatographic column was used with a flow rate of 12 mL / min, the mobile phase was 52% methanol, the detection wavelength of the UV detector was 368 nm, 500 μL was injected each time, and the chromatographic peak was collected for 30 min. After multiple accumulation, the target benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity was obtained by evaporation.

[0048] The application of the present invention is the application of the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity in the preparation of anti-methicillin-resistant Staphylococcus aureus (MRSA) drugs.

[0049] The present invention will be further described below with reference to specific examples of Cassia obtusifolia raw materials from different production areas in Yunnan:

[0050] Example 1

[0051] Preparation of benzo[g]chromone compounds C 16 H 14 O4, including extract extraction, silica gel column chromatography, and high pressure liquid chromatography separation, specifically adopts the following steps:

[0052] 1. Extraction of extract: Crush the whole plant of Cassia tora and use high concentration methanol (w%: 95%), high concentration ethanol (w%: 95%) or high concentration acetone (w%: 70%) as the extraction solvent, extraction solvent: whole plant of Cassia tora (weight ratio) = 3:5, soak for 54 hours, extract 4 times, combine the extracts, filter and concentrate into extract.

[0053] 2. Silica gel column chromatography: Dissolve the extract in 2.5 times the weight of pure methanol, pure ethanol or pure acetone, mix with 1.2 times the weight of 80-100 mesh silica gel, and dry-pack the column with 3 times the weight of 250 mesh silica gel for silica gel column chromatography; gradient elution is performed with chloroform-acetone solution with volume ratios of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2), combine the same fractions, collect the eluates from each fraction and concentrate.

[0054] 3. HPLC separation: The 7:3 portion of the column chromatography eluate was further separated and purified by HPLC to obtain the benzo[g]chromone compounds. The HPLC separation and purification was performed using a 21.2 mm × 250 mm, 5 μm C 18The chromatographic column was used, with a flow rate of 12 mL / min, a mobile phase of 52% methanol, a UV detector with a detection wavelength of 368 nm, 500 μL of sample was injected each time, and the chromatographic peak was collected for 30 min, and the sample was accumulated multiple times before evaporation to dryness.

[0055] A preferred post-processing scheme for the substance separated and purified by high performance liquid chromatography is to dissolve the obtained compound again in pure methanol and then separate it by gel column chromatography using pure methanol as the mobile phase for further separation and purification.

[0056] The whole plant of Cassia obovata used in the present invention is not limited by region or variety, and can be used to implement the present invention. The present invention is further described below using the whole plant of Cassia obovata from different production areas in Yunnan:

[0057] Example 2

[0058] The whole plant of Cassia obtusifolia was sourced from Yuanjiang, Yunnan. A 2.0 kg sample of the whole plant was crushed and extracted five times with 95% methanol, each extraction for 24 hours. The extracts were combined, filtered, and concentrated under reduced pressure to yield 82.5 g of extract. The extract was dissolved in 2.0 times the weight of pure methanol, mixed with 120 g of 100-mesh coarse silica gel, and then loaded onto a 0.6 kg 160-mesh silica column for silica gel column chromatography. Elution was performed using a chloroform-acetone gradient with a volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2. Identical fractions were combined and monitored by TLC to yield eight fractions. The fraction eluting with chloroform-acetone in a volume ratio of 7:3 was separated by semi-preparative HPLC on an Agilent 1100 with 52% methanol as the mobile phase and a Zorbax SB-C18 (21.2 × 250 mm, 5 μm) column. ） The preparative column was used as the stationary phase, the flow rate was 12 mL / min, the detection wavelength of the UV detector was 368 nm, 200 μL was injected each time, the chromatographic peak was collected for 30 minutes, and the chromatographic peak was accumulated multiple times and then evaporated to dryness; the obtained product was dissolved again in pure methanol and then separated by Sephadex LH-20 gel column chromatography using pure methanol as the mobile phase to obtain the new compound.

[0059] Example 3

[0060] The whole plant sample of Cassia obtusifolia was obtained from Hekou, Yunnan. A 3.5 kg sample of the whole plant was minced and extracted four times with 95% ethanol, each extraction time for 48 hours. The extracts were combined, filtered, and concentrated under reduced pressure to obtain 250 g of extract. The extract was dissolved in 2.0 times the weight of pure methanol and then mixed with 236 g of 80-mesh crude silica gel. 1.2 kg of 200-mesh silica gel was loaded onto a column for silica gel column chromatography. The elution was performed with a gradient of chloroform-acetone with a volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2. The same fractions were combined under TLC monitoring to obtain 8 fractions. The fraction eluted with chloroform-acetone with a volume ratio of 7:3 was separated by Agilent 1100 semi-preparative HPLC with 52% methanol as the mobile phase and a Zorbax SB-C18 (21.2 × 250 mm, 5 μm) preparative column as the stationary phase. The flow rate was 12 mL / min, the detection wavelength of the UV detector was 368 nm, 200 μL was injected each time, and 30 The chromatographic peak of min was accumulated multiple times and then evaporated to dryness; the obtained product was dissolved in pure methanol again and separated by Sephadex LH-20 gel column chromatography using pure methanol as the mobile phase to obtain the new compound.

[0061] Example 4

[0062] The whole plant of Cassia obtusifolia was obtained from Mengding, Yunnan. A 5 kg sample of the whole plant was crushed and ultrasonically extracted with 75% acetone three times, each extraction time for 72 hours. The extracts were combined, filtered, and concentrated under reduced pressure to yield 339 g of extract. The extract was dissolved in 1.6 times the weight of pure methanol and then mixed with 400 g of 90-mesh crude silica gel. The sample was loaded onto 2.4 kg of 180-mesh silica gel for silica gel column chromatography. The gradient elution was performed with chloroform-acetone ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2. The same fractions were combined under TLC monitoring to obtain 8 fractions. The fraction eluted with chloroform-acetone ratio of 7:3 was separated by Agilent 1100 semi-preparative HPLC with 52% methanol as the mobile phase and a Zorbax SB-C18 (21.2 × 250 mm, 5 μm) preparative column as the stationary phase. The flow rate was 12 ml / min, the detection wavelength of the UV detector was 368 nm, 200 μL was injected each time, and 30 The chromatographic peak of min was accumulated multiple times and then evaporated to dryness; the obtained product was dissolved in pure methanol again and separated by Sephadex LH-20 gel column chromatography using pure methanol as the mobile phase to obtain the new compound.

[0063] Example 5

[0064] Identification of compound structures

[0065] The structure of the benzo[g]chromone compounds prepared in Example 1-4 was determined by the following method. The compound of the present invention is a light yellow colloid. The compound of the present invention is a yellow colloid. 1 H and 13 C NMR spectrum (see Table 1, Figure 1 and Figure 2 ) showed that it contained 16 carbons and 14 hydrogens, including 1 tetrasubstituted naphthalene ring (C-5~C-13, C-4a, C-9a, H-5, H-7, H-8 and H-10), and 1 α,β -unsaturated carbonyl (-OC=CH-CO-, C-2~C-4, H-3), a methyl (C-16 and H3-16), a hydroxymethyl (C-15 and H2-15), and a methoxy ( δ C 56.1 and δ H 3.81 s). Further analysis of its NMR data, α,β The unsaturated carbonyl group is expected to connect to the naphthalene ring to form a pyran ring, forming a benzo[g]chromone skeleton, supporting the presence of ten degrees of unsaturation and two oxidized aromatic carbons (C-2 and C-3). This hypothesis is further confirmed by HMBC correlations between H-3 and C-2 / C-4 / C-12, H-5 to C-4 / C-6, H-7 to C-13, H-8 and C-14, and H-10 and C-9.

[0066] After the parent compound is determined, the remaining substituents (methoxy, methyl and hydroxymethyl) can be regarded as substituents on benzo[g]chromone. The methoxy substitution at the C-6 position can be achieved by methoxy hydrogen ( δ H 3.81 s) and C-6 were confirmed by HMBC correlation. HMBC correlations between H3-16 and C-8 / C-9 / C-14, and between H-8 and C-16, confirmed the methyl substitution at C-9. HMBC correlations between H2-15 and C-2 / C-3, and between H-3 and C-15, confirmed the hydroxymethyl substitution at C-2. Thus, the structure of the compound of the present invention was determined, and it was named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0067] Example 6

[0068] The compound prepared in Example 3 was a light yellow colloid. The determination method was the same as in Example 5, confirming that the compound prepared in Example 3 was the benzo[g]chromone compound. The compound was named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0069] Example 7

[0070] The compound prepared in Example 4 was a yellow colloid. The determination method was the same as that in Example 5, and the compound prepared in Example 4 was confirmed to be the compound described. The compound was named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0071] Example 8

[0072] The compounds of this invention were tested against drug-resistant bacteria. The test strain was a methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from clinical specimens at Kunming Medical University. The strain was positive for oxacillin resistance (≥4 mg / L) and the mecA gene was tested. Susceptibility testing was performed using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). The operating procedures were based on CLSIM07-A9 and the judgment criteria were based on CLSIM100-S24. The specific method is as follows:

[0073] (1) Preparation of Antimicrobial Stock Solution: The concentration of the antimicrobial stock solution is 2560 μg / mL. Antimicrobial drugs with low solubility may be slightly lower than this concentration. The required amount of antimicrobial solution or powder can be calculated using the formula. The prepared antimicrobial stock solution should be stored below -20°C and has a shelf life of no more than 6 months.

[0074] (2) Preparation of test bacteria: Use an inoculation loop to pick a single colony from the overnight cultured MH(A) dish and place it in MH(B) medium, calibrate it to a 0.5 McFarland turbidimetric standard, containing approximately 1×108 CFU / ml of bacteria, and then dilute it 100-fold to obtain a culture medium containing approximately 1×10 6 CFU / mL of bacterial solution for future use.

[0075] (3) Dilute the stock solution of antimicrobial drugs (2560 μg / mL) 10 times to obtain an antimicrobial drug solution with a concentration of 256 μg / mL. Take a sterile 96-well plate, add 200 μL of antimicrobial drugs to the first well, add 100 μL of MH broth culture medium to the second to tenth wells, draw 100 μL from the first well and add it to the second well, mix well, draw 100 μL to the third well, and so on. Draw 100 μL from the tenth well and discard. At this time, the drug concentrations in each well are: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / mL, add 100 μL of bacterial solution to the eleventh well (positive control), and add 100 μL of MH(B) culture medium to the twelfth well (negative control).

[0076] (4) Add 100 μL of the previously prepared bacterial solution to each well from 1 to 10, so that the final bacterial solution concentration in each tube is approximately 5×10 5 CFU / mL, with drug concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL in wells 1 to 10, respectively. Place the inoculated 96-well plate in a 37°C incubator and observe bacterial growth for 24 hours. Use a standard strain for quality control.

[0077] (5) Result evaluation and interpretation: Before reading and reporting the MIC of the test strain, the growth control tube should be checked for good bacterial growth. The inoculum subculture should also be checked to determine whether it is contaminated and whether the MIC value of the quality control strain is within the quality control range. The tube with the lowest drug concentration that shows no bacterial growth by visual observation is the MIC of the test strain.

[0078] (6) The test results showed that compound (1) has outstanding antibacterial activity and has obvious antibacterial advantages compared with the antibacterial drugs used in clinical treatment of Gram-positive bacteria infections. 90 The concentration was 52.84±4.2 μg / mL, with significant antibacterial activity, close to the MIC of levofloxacin. 90 value (>50.84 ± 5.5 μg / mL).

[0079] Example 9

[0080] The compounds of the present invention were subjected to toxicological evaluation, and the mouse bone marrow micronucleus test, Ames test and TK gene mutation test all proved that the compounds of the present invention were non-toxic to animals and safe to use.

Claims

1. A benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, characterized in that: The benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus activity is prepared from the whole plant of the medicinal plant Cassia torafolia by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. It is named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4 H -Chromone, English name: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4 H -chromone, with the following structure: 。 2. A method for preparing the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity according to claim 1, characterized in that: The preparation method is prepared from the whole plant of the medicinal plant Cassia torafolia as raw material through pretreatment, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically includes the following steps: A. Pretreatment: crush or cut the whole plant of the raw medicinal plant Cassia tora into segments to obtain material a; B. Organic solvent extraction: Add 2-4 times the mass of organic extraction solvent to material a, soak and extract 3-5 times at room temperature, each extraction time is 24-72 hours, combine the extracts, filter and concentrate to obtain extract b; C. Silica gel column chromatography: Add 160-300 mesh silica gel in an amount 2-4 times the weight of extract b to the column and dry-pack it. Use a chloroform-acetone solution with a volume ratio of 1:0-1:2 for gradient elution. Monitor by TLC and combine the same fractions. D. High Performance Liquid Chromatography Separation: The eluate obtained by eluting with a chloroform-acetone solution in a ratio of 7:3 was separated and purified by high performance liquid chromatography to obtain the target benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus activity.

3. The preparation method according to claim 2, characterized in that The organic extraction solvent in step B is an aqueous methanol solution with a mass concentration of 80% to 100%, an aqueous ethanol solution with a mass concentration of 80% to 100%, or an aqueous acetone solution with a mass concentration of 60% to 90%.

4. The preparation method according to claim 2, characterized in that Step C also includes the step of dissolving the extract b with an organic solvent at a volume of 1.5 to 3 times the weight of the extract b and then adding 80 to 100 mesh silica gel at a volume of 0.8 to 1.2 times the weight of the extract b to mix the sample before loading onto the column.

5. The preparation method according to claim 2, characterized in that The organic extraction solvent is pure methanol, pure ethanol or pure acetone.

6. The preparation method according to claim 2, characterized in that The volume ratios of the chloroform-acetone solution described in step C are 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:

2.

7. The preparation method according to claim 2, characterized in that The high-pressure liquid chromatography separation and purification in step D was performed using a 21.2 mm × 250 mm, 5 μ m's C 18 The column was chromatographically loaded with a flow rate of 12 mL / min, the mobile phase was 52% methanol, the detection wavelength of the UV detector was 368 nm, and 500 μL of sample was injected each time. μ L, collect the chromatographic peak for 30 min, accumulate it multiple times and evaporate to dryness to obtain the target benzo[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus activity.

8. Use of the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity according to claim 1, characterized in that: The invention relates to an application of the benzo[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus activity in the preparation of an anti-methicillin-resistant Staphylococcus aureus drug.

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