CN107815473B - Diphenyl ether compound and preparation method and application thereof - Google Patents
Diphenyl ether compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a diphenyl ether compound and a preparation method and application thereof. The diphenyl ether compound is obtained by fermenting, extracting, chromatographing and purifying Phomopsis fukushii strain, and the molecular formula of the diphenyl ether compound is C18H20O5The structural formula is as follows:
the compound was named: diphenther B; the strain of Phomopsis Fukushii has the preservation number of CCTCC M2017632 and the preservation date of 10 months and 23 days in 2017. The preparation method takes a solid fermentation product of Phomopsis fukushii strain as a raw material, and comprises the steps of organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation. The application is the application of the diphenyl ether compound in preparing a methicillin-resistant staphylococcus aureus medicament. The compound has the advantages of novel structure, good activity and good application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a diphenyl ether compound and a preparation method and application thereof.
Background
In 1978, medical staff randomly examined 200 strains of staphylococcus aureus in Shanghai, and the number of separated methicillin-resistant staphylococcus aureus (MRSA) was less than 5%. A sampling survey of hospitalized patients in 2009 showed that methicillin-resistant staphylococcus aureus (MRSA) was over 60%, increasing by 40% compared to 2000. The DNA of the intestinal flora of Chinese, Denmark and Spain was sequenced by Zhu Bao Li and co-workers at the institute of microbiology, university of Chinese medical science, and the study showed that: chinese intestinal flora has more drug-resistant genes. Therefore, the research and development of new strategies and new drugs capable of effectively controlling drug-resistant bacterial infection become a hotspot of antibiotic research.
Disclosure of Invention
The first purpose of the invention is to provide a diphenyl ether compound; the second purpose is to provide a preparation method of the diphenyl ether compound; the third purpose is to provide the application of the diphenyl ether compound.
The first purpose of the invention is realized by that the diphenyl ether compound is obtained by fermenting, extracting, chromatographing and purifying Phomopsis fukushi strain, and the molecular formula of the diphenyl ether compound is C18H20O5The structural formula is as follows:
the compound was named: diphenther B;
the strain of Phomopsis Fukushii has the preservation number of CCTCC M2017632 and the preservation date of 10 months and 23 days in 2017.
The second object of the present invention is achieved by comprising the steps of:
A. solid fermentation: culturing a strain Phomopsis fukushii in a potato glucose agar culture medium at 28 ℃ for 7 days, inoculating the strain to a 50-500 mL triangular flask containing 10-100 mL of a liquid seed culture medium, performing shake culture at 28 ℃ for 5-10 days under the condition of rotary shake at a rotating speed of 180rpm to obtain a bacterium-containing nutrient medium, and inoculating the bacterium-containing nutrient medium to a rice solid culture medium to perform culture to obtain a fermentation product;
B. extracting the extractum: adding an organic solvent with the solid-liquid volume ratio of 1.5-3 times into the fermentation product, performing ultrasonic extraction for 2-4 times, each time for 4-8 hours, combining the extracting solutions, filtering, performing reduced pressure concentration until the volume is 1/4-1/2, standing, filtering out precipitates, and concentrating to obtain an extract;
C. chloroform-acetone silica gel column chromatography: performing silica gel column chromatography on the extract, wherein silica gel filled in the column is 200-300 meshes, and the using amount of the silica gel is 6-8 times of the weight of the extract; performing gradient elution by using chloroform and/or acetone solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. silica gel column chromatography with petroleum ether-ethyl acetate: performing silica gel column chromatography on an eluent obtained by eluting the chloroform-acetone mixed solution with the ratio of 8:2 in the step C, loading the silica gel into a column with 200-300 meshes, using the amount of the silica gel being 6-8 times of the weight of the eluent, performing gradient elution by using petroleum ether and/or ethyl acetate solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
E. high-pressure liquid chromatography separation and purification: eluting the petroleum ether-ethyl acetate mixed solution with the ratio of 7:3 in the step D to obtain an eluent, taking 40-60% of methanol as a mobile phase, and taking C with the specification of 20mm multiplied by 250mm and 5 mu m18The preparation column is a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 284nm, 200 mu L of eluent is injected each time, eluent is collected and is evaporated to dryness after being accumulated for multiple times, and the diphenther B is obtained.
The structure of the diphenyl ether compound of the present invention is determined by the following method:
the compound of the invention is light yellow oil; ultraviolet spectrum (the solvent is methanol),λ max（logε) 287 (3.60), 206 (4.63); infrared spectrum (Potassium bromide tablet)ν max:3450，2939，2834，1686，1615，1459，1347，1164，1055，872cm-1(ii) a HRESIMS shows the molecular ion peak M/z339.1216[ M + Na ] of the compound of the invention plus sodium]+（C18H20NaO5Calculated 339.1208), combined1H and13the C NMR spectrum (FIGS. 2 and 3, data retention in Table 1) gives the formula C18H20O5。
Process for preparing compounds1The H NMR spectrum shows that the compound has 19 hydrogen signals including a methyl proton signal H3-7′（d H2.27 s), one hydroxymethyl proton signal H2-7（d H4.64 s), five aryl proton signals [ H-2 ] (d H6.47s），H-4（d H6.57s），H-6（d H6.50s），H-2'（d H6.37d J=2.2），H-6'（d H6.31d J=2.2]Two methoxy signals [ OMe-3 ] (d H3.81s），OMe-3′（d H3.86s）]One acetyl proton signal H3-9'（d H2.58 s). Process for preparing compounds13The C NMR spectrum showed 18 carbon signals in the compound, including a methyl carbon signal C-7' (d)C23.5 q), one hydroxymethyl carbon signal C-7 (d)C68.2 q), two methoxy carbon signals [3-OMe (d)C55.9q），3＇-OMe（dC56.2q）]The acetyl carbon signal [ C-8' ("dC198.7s），C-9＇（dC29.8q）]Five sp2Hybridized aryl methine carbon Signal [ C-2 (d)C104.4d），C-4（dC106.6d），C-6（dC108.6d），C-2＇（dC100.5d），C-6＇（dC113.6d）]Seven sp2Hybrid aryl quaternary carbon Signal [ C-1 (d)C155.1s），C-3（dC162.8s），C-5（dC143.2s），C-1＇（dC162.3s），C-3＇（dC164.1s），C-4＇（dC110.4s），C-5＇（dC142.3s）]Wherein four carbons are linked to oxygen. Synthesis of1H NMR spectrum and13c NMR spectrum data show that the compound has two benzene rings. There are four oxygen-linked quaternary carbons in the two phenyl rings, except for two methoxy substitutions, one oxygen atom should be attached to both phenyl rings to form an ether linkage. The compound is diphenyl ether derivative with one methyl group, one hydroxymethyl group, two methoxy groups and one acetyl group in the benzene ringAnd (4) substituting the group. The infrared spectrum showed hydroxyl (3450 cm)-1) Carbonyl group (1686 cm)-1) And phenyl (1615, 1459, 1347 cm)-1) The absorption peak of (1). H-9' (II) in HMBC spectraδ H2.58) and C-4' ((II)δ C110.4) correlation, confirming the attachment of the acetyl group at the C-4' position, methoxy proton OMe: (δ H3.81) and C-3 (δ C162.8), methoxy proton OMe (A), (B), (C), (δ H3.86) and C-3' ((III)δ C164.1) correlation, confirming that the two methoxy groups are respectively connected at the C-3 and C-3' positions, and the methyl proton H3-7′（δ H2.27) and C-4' ((II)δ C110.4）/C-5′（δ C142.3）/C-6′（δ C113.6) and confirming the attachment of the methyl group at the C-5' position. Hydroxymethyl proton Signal H2-7（d H4.64) and C-4 (Cδ C106.6）/C-5（δ C143.2）/C-6（δ C108.6) correlation, confirming the attachment of the hydroxymethyl group at the C-5 position. In addition, the characteristic proton signal H-2 (delta)H6.47s），H-4（δH6.57s），H-6（δH6.50，s），H-2′（δH6.37, d, J = 2.2) and H-6' (δH6.31, d, J = 2.2) also demonstrate the position of the above substituents, and therefore the structure of the compound is confirmed.
Of the compounds of Table 11H and13c NMR data (solvent C)5D5N）
The third purpose of the invention is realized by the application of the diphenyl ether compound in preparing medicines for resisting methicillin-resistant staphylococcus aureus.
In recent years, the focus of research has turned back to the search for new secondary skeletal metabolites with different antibacterial mechanisms from microorganisms, and endophytic fungal metabolites are the most attractive hot spots in this field, where the group most frequently producing antibacterial active compounds is the phoiomimeticThe genera Phomopsis (Phomopsis), Phoma (Phoma) and Fusarium (Fusarium). The phomopsis is a large genus of fungi of Deuteromycotina and coelomycetes, and the strain can produce rich secondary metabolite, and Weber and the like are separated from erythrina cristataPhomopsis sp. to obtain a new antibacterial activity lactone compound phomol, which shows good antibacterial activity to 24 bacteria and fungi by biological tests. Horn et al isolated from Salix integraPhomopsis sp.The strain and the separated cytochalasin alkaloid phomopsichalasin can inhibit staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, staphylococcus aureus and escherichia coli.
Many natural diphenyl ethers exhibit excellent antibacterial activity. The invention relates to a phomopsis Fuji strainPhomopsis fukushii) Separating a new diphenyl ether compound diphenther B from the fermentation product, wherein the compound has obvious MRSA (methicillin-resistant Staphylococcus aureus) activity.
The diphenther B is separated for the first time, and the molecular formula and the structure of the diphenther B are determined by nuclear magnetic resonance, mass spectrum, infrared spectrum and ultraviolet spectrum data. The activity of the staphylococcus aureus resistant standard strain ATCC25923 and the methicillin resistant standard strain ATCC43300 is tested by a trace broth dilution method, the MIC of the diphenther B capable of resisting the staphylococcus aureus resistant standard strain ATCC25923 is 8 mu g/mL, and the MIC of the MRSA resistant standard strain ATCC43300 is 8 mu g/mL, so that the compound has better MRSA resistance activity and can be used as a lead compound for resisting MRSA.
Drawings
FIG. 1 shows the NMR spectrum of a compound (A)1H NMR）；
FIG. 2 is a nuclear magnetic resonance carbon spectrum of a compound (C13C NMR）；
Figure 3 is an HMBC correlation spectrum of a compound.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The diphenyl ether compound is obtained by fermenting, extracting, chromatographing and purifying Phomopsis fukushii strain, and the molecular formula of the diphenyl ether compound is C18H20O5The structural formula is as follows:
the compound was named: diphenther B;
the strain of Phomopsis Fukushii has the preservation number of CCTCC M2017632 and the preservation date of 10 months and 23 days in 2017.
The preparation method of the diphenyl ether compound comprises the following steps:
A. solid fermentation: culturing a strain Phomopsis fukushii in a potato glucose agar culture medium at 28 ℃ for 7 days, inoculating the strain to a 50-500 mL triangular flask containing 10-100 mL of a liquid seed culture medium, performing shake culture at 28 ℃ for 5-10 days under the condition of rotary shake at a rotating speed of 180rpm to obtain a bacterium-containing nutrient medium, and inoculating the bacterium-containing nutrient medium to a rice solid culture medium to perform culture to obtain a fermentation product;
B. extracting the extractum: adding an organic solvent with the solid-liquid volume ratio of 1.5-3 times into the fermentation product, performing ultrasonic extraction for 2-4 times, each time for 4-8 hours, combining the extracting solutions, filtering, performing reduced pressure concentration until the volume is 1/4-1/2, standing, filtering out precipitates, and concentrating to obtain an extract;
C. chloroform-acetone silica gel column chromatography: performing silica gel column chromatography on the extract, wherein silica gel filled in the column is 200-300 meshes, and the using amount of the silica gel is 6-8 times of the weight of the extract; performing gradient elution by using chloroform and/or acetone solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. silica gel column chromatography with petroleum ether-ethyl acetate: performing silica gel column chromatography on an eluent obtained by eluting the chloroform-acetone mixed solution with the ratio of 8:2 in the step C, loading the silica gel into a column with 200-300 meshes, using the amount of the silica gel being 6-8 times of the weight of the eluent, performing gradient elution by using petroleum ether and/or ethyl acetate solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
E. high-pressure liquid chromatography separation and purification: eluting the petroleum ether-ethyl acetate mixed solution with the ratio of 7:3 in the step D to obtain an eluent, taking 40-60% of methanol as a mobile phase, and taking C with the specification of 20mm multiplied by 250mm and 5 mu m18The preparation column is a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 284nm, 200 mu L of eluent is injected each time, eluent is collected and is evaporated to dryness after being accumulated for multiple times, and the diphenther B is obtained.
The potato glucose agar culture medium in the step A is prepared by 200g/L of potatoes, 20g/L of glucose, 20g/L of agar, 6.5 of pH value, sterilization at 121 ℃ for 25min, and cooling to 60 ℃ to prepare an inverted slope.
The liquid seed culture medium in the step A is NaNO3 2g/L，K2HPO4 1g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H2O0.01 g/L, glucose 20g/L, pH 6.5, and sterilizing at 121 deg.C for 25min in a 250mL conical flask or 100 mL/flask.
The rice solid culture medium in the step A is prepared by filling 100g of rice, 20g of perlite and 100mL of distilled water into a 600mL tissue culture bottle, and sterilizing at 121 ℃ for 25 min.
And the organic solvent in the step B is acetone, ethanol or methanol with the volume concentration of 60-80%.
And C, before the extractum is subjected to silica gel column chromatography, dissolving the extractum by using acetone with the weight ratio of 1.5-3 times, and then mixing the extractum by using 200-300-mesh silica gel with the weight ratio of 1-3 times.
And C, the volume ratio of the chloroform and/or acetone solution in the step C is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0: 1.
The volume ratio of the petroleum ether and/or ethyl acetate solution in the step D is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0: 1.
The preparation method of the diphenyl ether compound provided by the invention specifically comprises the following operations:
(1) placing the phomopsis Fuji in a slant solid culture medium for storage for later use, transferring the strain for storage for later use into a liquid seed culture medium, and culturing for 24h at 28 ℃ on a shaking bed at the rotating speed of 180r/min to obtain a phomopsis Fuji seed solution; and (3) taking the phomopsis formosana seed liquid, inoculating the phomopsis formosana seed liquid into a rice solid culture medium according to the mass ratio of 2-5% of the culture medium, and fermenting for 30-60 days at the temperature of 25-35 ℃ to obtain a fermentation product.
(2) Ultrasonic extraction: taking 60-80% acetone as a solvent, performing ultrasonic extraction for 3-5 times, each time for 4-8 hours, combining the extracting solutions, filtering, and concentrating under reduced pressure to obtain an extract;
(3) chloroform-acetone silica gel column chromatography: dissolving the extract by using 1.5-3 times of pure acetone by weight, mixing the extract by using 1-3 times of 200-mesh silica gel and 300-mesh silica gel, loading the mixture into a silica gel column, performing gradient washing by using chloroform and/or acetone solutions with volume ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0:1, respectively collecting and concentrating the eluates of each part, and respectively collecting and concentrating the eluates of each part;
(4) performing petroleum ether-ethyl acetate silica gel column chromatography, namely dissolving the fraction 8:2 in the step (3) by using pure methanol with the weight ratio of 1.5-3 times, mixing the sample by using 200-mesh 300-mesh silica gel with the weight of 1-3 times that of the extract, putting the mixture into a silica gel column, performing gradient washing by using petroleum ether and/or ethyl acetate solutions with the volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0:1, and respectively collecting and concentrating the eluates of each part;
(5) high-pressure liquid chromatography separation: taking 40-60% methanol of the fraction in the step (4) 7:3 as a mobile phase, and taking C with the specification of 20mm multiplied by 250mm and 5 mu m18The preparation column is a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 284nm, 200 mu L of eluent is injected each time, the eluent is collected, and the diphenther B is obtained after the eluent is evaporated to dryness after being accumulated for multiple times.
The diphenyl ether compound is applied to the preparation of a methicillin-resistant staphylococcus aureus medicament.
The invention is further illustrated by the following specific examples:
example 1
(1) The phomopsis Fuji CCTCC M2017632 preserved in the slant solid culture medium is inoculated in a plate of the slant solid culture medium for activation, and the phomopsis Fuji CCTCC M2017632 is inoculated in the slant solid culture medium for storage for later use after purification. The slant solid culture medium is as follows: 200g/L of potato, 20g/L of glucose, 20g/L of agar, 6.5 of pH value, 121 ℃, 25min of sterilization, cooling to 60 ℃, and pouring a flat plate and a bevel for standby;
(2) and (3) transferring the preserved phomopsis formosanus CCTCC M2017632 into a liquid seed culture medium, and culturing for 24h at 28 ℃ on a shaking bed at the rotating speed of 180r/min to obtain the phomopsis formosanus seed liquid. The liquid seed culture medium is as follows: NaNO3 2g/L，K2HPO4 1g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H20.01g/L of O, 20g/L of glucose and 6.5 of pH value, and the mixture is filled into a 250mL conical flask and a 100mL conical flask, sterilized for standby at 121 ℃ for 25 min;
(3) and (3) inoculating the phomopsis formosana seed liquid obtained in the step (2) into a rice solid culture medium according to the mass ratio of 2% of the culture medium for solid fermentation, wherein the fermentation temperature is 25 ℃, and the fermentation is completed within 45 days. The rice culture medium is as follows: 100g of rice, 20g of perlite and 100mL of distilled water are put into a 600mL tissue culture bottle and sterilized for standby at 121 ℃ for 25 min.
Example 2
Example 1 was repeated with the following differences:
(3) the fermentation temperature is 28 ℃, and the fermentation is completed in 40 days.
Example 3
(3) The fermentation temperature is 30 ℃, and the fermentation is completed in 30 days.
Example 4
(3) And inoculating the phomopsis Fuji seed liquid into a rice solid culture medium according to the mass ratio of 5% of the culture medium for solid fermentation, wherein the fermentation temperature is 28 ℃, and the fermentation is completed within 35 days.
Example 5
(1) Taking 10kg of fermentation product of Phomopsis Fuji CCTCC M2017632, ultrasonically extracting with 80% acetone for 4 times, each time for 4h, mixing the extractive solutions, filtering, and concentrating under reduced pressure to obtain 562g of extract.
(2) Dissolving the extract with 1000mL of acetone, mixing with 200g of 200-mesh 300-mesh silica gel, loading into 1200g of 200-mesh 300-mesh silica gel column for chromatographic separation, performing gradient elution by using chloroform-acetone solutions with volume ratios of 20:1, 9:1 and 8:2, collecting eluates of each part respectively, and concentrating.
(3) Taking 56.3g of chloroform-methanol 8:2 elution concentration part in the step (2), dissolving with 120mL of pure methanol, mixing with 200-mesh 300-mesh silica gel of which the weight is 1-3 times that of the extract, loading on a silica gel column, washing with petroleum ether and/or ethyl acetate solution in a volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0:1 in a gradient manner, collecting eluates of each part respectively and concentrating;
(4) taking 12.5g of fraction 7:3 in step (3), using 56% methanol as mobile phase, and C with specification of 20mm × 250mm and 5 μm18The preparation column is a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 287nm, 200 mu L of sample is injected each time, a chromatographic peak of 15.1min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation, so that the diphenther B disclosed by the invention is obtained.
Example 6
Example 5 was repeated with the following differences:
(1) ultrasonically extracting with 70% acetone for 4 times, each time for 5 hr; 622g of extract A was concentrated under reduced pressure.
(4) The petroleum ether-ethyl acetate 7:3 eluate fraction was 15.6g, and high performance liquid chromatography was performed using 50% methanol as the mobile phase to collect the chromatographic peak at 13.8 min.
Example 7
Example 5 was repeated with the following differences:
(1) ultrasonic extracting with 75% acetone for 3 times; the extract A obtained by vacuum concentration was 577 g.
(4) The petroleum ether-ethyl acetate 7:3 elution fraction was 13.3g, and a chromatographic peak was collected for 11.2min with 60% methanol as the mobile phase.
Example 8
Example 5 was repeated with the following differences:
(4) the petroleum ether-ethyl acetate 7:3 eluate fraction was 11.6g, and high performance liquid chromatography was performed using 50% methanol as the mobile phase to collect the 18.3min chromatographic peak.
Example 9
The compound prepared in example 5 was taken as a pale yellow gum and determined by the following method: nuclear magnetic resonance, in combination with other spectroscopic techniques, was used to identify structures.
(1) Ultraviolet spectrum (the solvent is methanol),λ max（logε）:287（3.60），206（4.63）；
(2) infrared spectrum (Potassium bromide tablet)ν max:3450，2939，2834，1686，1615，1459，1347，1164，1055，872cm-1(ii) a HRESIMS shows the molecular ion peak M/z339.1216[ M + Na ] of the compound of the invention plus sodium]+（C18H20NaO5Calculated 339.1208), combined1H and13the C NMR spectrum (FIGS. 2 and 3, data retention in Table 1) gives the formula C18H20O5。
Process for preparing compounds1The H NMR spectrum shows that the compound has 19 hydrogen signals including a methyl proton signal H3-7′（d H2.27 s), one hydroxymethyl proton signal H2-7（d H4.64 s), five aryl proton signals [ H-2 ] (d H6.47s），H-4（d H6.57s），H-6（d H6.50s），H-2'（d H6.37d J=2.2），H-6'（d H6.31d J=2.2]Two methoxy signals [ OMe-3 ] (d H3.81s），OMe-3′（d H3.86s）]One acetyl proton signal H3-9'（d H2.58 s). Process for preparing compounds13The C NMR spectrum showed 18 carbon signals in the compound, including a methyl carbon signal C-7' (d)C23.5 q), one hydroxymethyl carbon signal C-7 (d)C68.2 q), two methoxy carbon signals [3-OMe (d)C55.9q），3＇-OMe（dC56.2q）]The acetyl carbon signal [ C-8' ("dC198.7s），C-9＇（dC29.8q）]Five sp2Hybridized aryl methine carbon Signal [ C-2 (d)C104.4d），C-4（dC106.6d），C-6（dC108.6d），C-2＇（dC100.5d），C-6＇（dC113.6d）]Seven sp2Hybrid aryl quaternary carbon Signal [ C-1 (d)C155.1s），C-3（dC162.8s），C-5（dC143.2s），C-1＇（dC162.3s），C-3＇（dC164.1s），C-4＇（dC110.4s），C-5＇（dC142.3s）]Wherein four carbons are linked to oxygen. Synthesis of1H NMR spectrum and13c NMR spectrum data show that the compound has two benzene rings. There are four oxygen-linked quaternary carbons in the two phenyl rings, except for two methoxy substitutions, one oxygen atom should be attached to both phenyl rings to form an ether linkage. The compound is diphenyl ether derivative, and a methyl group, a hydroxymethyl group, two methoxyl groups and an acetyl group are substituted on a benzene ring. The infrared spectrum showed hydroxyl (3450 cm)-1) Carbonyl group (1686 cm)-1) And phenyl (1615, 1459, 1347 cm)-1) The absorption peak of (1). H-9' (II) in HMBC spectraδ H2.58) and C-4' ((II)δ C110.4) correlation, confirming the attachment of the acetyl group at the C-4' position, methoxy proton OMe: (δ H3.81) and C-3 (δ C162.8), methoxy proton OMe (A), (B), (C), (δ H3.86) and C-3' ((III)δ C164.1) correlation, confirming that the two methoxy groups are respectively connected at the C-3 and C-3' positions, and the methyl proton H3-7′（δ H2.27) and C-4' ((II)δ C110.4）/C-5′（δ C142.3）/C-6′（δ C113.6) and confirming the attachment of the methyl group at the C-5' position. Hydroxymethyl proton Signal H2-7（d H4.64) and C-4 (Cδ C106.6）/C-5（δ C143.2）/C-6（δ C108.6) correlation, confirming the attachment of the hydroxymethyl group at the C-5 position. In addition, the characteristic proton signal H-2 (delta)H6.47s），H-4（δH6.57s），H-6（δH6.50，s），H-2′（δH6.37, d, J = 2.2) and H-6' (δH6.31, d, J = 2.2) also demonstrate the position of the above substituents, and therefore the structure of the compound is confirmed.
Example 10
The compounds prepared in examples 6, 7 and 8 were takenAll of which were yellow gels, and the determination method was the same as in example 9, and all of which confirmed that the compounds prepared in examples 6, 7 and 8 were C18H20O5。
Example 11
An activity detection test is carried out on any compound prepared in the embodiment 5-8, and the test conditions are as follows:
the MIC (. mu.g/mL) value of diphenther B was determined using the broth dilution method.
(1) Preparing an antibacterial agent and a culture medium: dissolving the diphenyl ether compound to be detected in DMSO to prepare mother liquor with the concentration (2560 mu g/mL), and filtering and sterilizing the mother liquor for later use. The prepared MH broth culture medium is sterilized for 30min at 121 ℃ for standby.
(2) The MRSA colonies cultured for 24 hours were prepared into a suspension of 0.5 M.turbidimetric standard. Diluting the bacterial suspension with MH broth 1: 100 to obtain about 1 × 10 bacteria6CFU/mL of the bacterial liquid for later use.
(3) Preparation of diluted antibacterial agent and inoculation of bacterial solution 13 sterile test tubes (13X 100 mm) are taken, except that 1.6mL of MH broth is added into the 1 st tube, 1mL of MH broth is added into each tube, 0.4mL of antibacterial agent stock solution (such as 1280 mu g/mL) is added into the 1 st tube and mixed evenly, then 1mL is sucked into the 2 nd tube, 1mL is sucked into the 3 rd tube after mixing evenly, dilution is carried out to the 11 th tube in a multiple ratio in this way continuously, 1mL is sucked from the 11 th tube and discarded, and the 12 th tube is a growth control without the medicine. At this time, the drug concentration in each tube was 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25. mu.g/mL in this order. Then 1mL of the prepared inoculum is added into each tube, the drug concentration of the 1 st tube to the 11 th tube is respectively 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125 mu g/mL, the culture in the dilution tube is transferred into a 96-well plate, each sample is divided into three parallels, DMSO is used as a solvent control, and vancomycin is used as a positive control.
(4) Incubation the inoculated 96-well plate was incubated in a 37 ℃ incubator for 24 h.
(5) And (5) judging a result: the lowest drug concentration that completely inhibited bacterial growth in the wells was the MIC, while there was no significant inhibition of bacteria in the solvent control.
The strains used in the broth microdilution method include Staphylococcus aureus standard strain ATCC25923, methicillin-resistant Staphylococcus aureus standard strain ATCC 43300. The result shows that the diphenther B can inhibit ATCC25923 and ATCC43300, the MIC of the staphylococcus aureus standard strain ATCC25923 is 8 mu g/mL, the MIC of the methicillin-resistant staphylococcus aureus standard strain ATCC43300 is 8 mu g/mL, and the diphenther B can be used as a lead compound for developing methicillin-resistant staphylococcus aureus medicaments.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and changes made within the spirit and principles of the present invention are intended to be included therein.
Claims (8)
1. A process for preparing diphenyl ether compounds, which is characterized by that the diphenyl ether compounds are phomopsis Fushii: (A)Phomopsis fukushii) The strain is obtained by fermenting, extracting, chromatography and purifying, and has molecular formula of C18H20O5The structural formula is as follows:
the compound was named: diphenther B; said phomopsis formosana (A), (B), (C), (Phomopsis fukushii) The strain has a preservation number of CCTCC M2017632 and a preservation date of 2017, 10 months and 23 days; the preparation method comprises the following steps:
A. solid fermentation: the strain Phomopsis Fuji (Phomopsis fukushii) Culturing for 7 days at 28 ℃ in a potato glucose agar culture medium, inoculating the potato glucose agar culture medium into a 50-500 mL triangular flask containing 10-100 mL liquid seed culture medium, performing shake culture for 5-10 days at 28 ℃ under the condition of rotary shake at the rotating speed of 180rpm to obtain a bacterium-containing nutrient medium, and inoculating the bacterium-containing nutrient medium into a rice solid culture medium to perform culture to obtain a fermentation product;
B. extracting the extractum: adding an organic solvent with the solid-liquid volume ratio of 1.5-3 times into the fermentation product, performing ultrasonic extraction for 2-4 times, each time for 4-8 hours, combining the extracting solutions, filtering, performing reduced pressure concentration until the volume is 1/4-1/2, standing, filtering out precipitates, and concentrating to obtain an extract;
C. chloroform-acetone silica gel column chromatography: performing silica gel column chromatography on the extract, wherein silica gel filled in the column is 200-300 meshes, and the using amount of the silica gel is 6-8 times of the weight of the extract; performing gradient elution by using chloroform and/or acetone solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. silica gel column chromatography with petroleum ether-ethyl acetate: performing silica gel column chromatography on an eluent obtained by eluting the chloroform-acetone mixed solution with the ratio of 8:2 in the step C, loading the silica gel into a column with 200-300 meshes, using the amount of the silica gel being 6-8 times of the weight of the eluent, performing gradient elution by using petroleum ether and/or ethyl acetate solution with the volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
E. separating and purifying by high performance liquid chromatography: eluting the petroleum ether-ethyl acetate mixed solution with the ratio of 7:3 in the step D to obtain an eluent, taking 40-60% of methanol as a mobile phase, and taking C with the specification of 20mm multiplied by 250mm and 5 mu m18The preparation column is a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 284nm, 200 mu L of eluent is injected each time, eluent is collected and is evaporated to dryness after being accumulated for multiple times, and the diphenther B is obtained.
2. The method according to claim 1, wherein the potato dextrose agar medium in step A is prepared by sterilizing potato at 200g/L, glucose at 20g/L, agar at 20g/L, pH 6.5, 121 ℃, for 25min, and cooling to 60 ℃ on a reverse slope.
3. The method according to claim 1, wherein the liquid seed medium in step A is NaNO32g/L，K2HPO4 1g/L，MgSO4·7H2O 0.5g/L，KCL 0.5g/L，FeSO4·7H2O0.01 g/L, glucose 20g/L, pH 6.5, and sterilizing at 121 deg.C for 25min in a 250mL conical flask or 100 mL/flask.
4. The preparation method of claim 1, wherein the rice solid medium in step A is prepared from rice 100g, perlite 20g, and distilled water 100mL by placing into 600mL tissue culture bottle, sterilizing at 121 deg.C for 25 min.
5. The method according to claim 1, wherein the organic solvent in step B is acetone, ethanol or methanol with a volume concentration of 60-80%.
6. The preparation method according to claim 1, wherein the extract in step C is dissolved in 1.5-3 times of acetone before being subjected to silica gel column chromatography, and then is mixed with 1-3 times of 200-300 mesh silica gel.
7. The method according to claim 1, wherein the volume ratio of the chloroform and/or acetone solution in step C is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0: 1.
8. The method according to claim 1, wherein the volume ratio of the petroleum ether and/or ethyl acetate solution in step D is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0: 1.
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| CN103589757A (en) * | 2013-02-04 | 2014-02-19 | 中国海洋大学 | Preparation method for bromo-diphenyl ether derivatives and applications as antibacterial agents |
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