Use of diphenyl ether compounds for the preparation of beta-glucuronidase inhibitors

Abstract

The application discloses application of a diphenyl ether compound in preparation of a beta-glucuronidase inhibitor. The diphenyl ether compound is obtained from a fungus Aspergillus versicolor ZJUTE2 through a series of processes such as organic solvent extraction and chromatographic column separation, simple extraction and separation, and has the advantages of simple preparation method, rapidness, high purity of obtained compound and the like; the diphenyl ether compound extracted by the application has good beta-glucuronidase inhibitory activity, and an IC 50 value of 56.59+ / -1.79 muM, is equivalent to a positive drug D-glucaric acid 1,4-lactone, and can be used for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs. In addition, development of structure optimization based on the compound as a lead is of great significance for research and development of a novel drug for treating drug-induced diarrhea.

Description

(I)TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an application of a diphenyl ether compound 7-hydroxy-diorcinol isolated from a fungus Aspergillus versicolor ZJUTE2 in inhibiting activity of beta-glucuronidase from Escherichia coli. (II)BACKGROUND

[0002] Glucuronidation is one of the most important detoxification processes in the human body, and most endogenous substances and drugs can be transported to the duodenum through a single tube after glucuronidation, and then hydrolyzed by beta-glucuronidase secreted by intestinal flora to form aglycone, and then the free aglycone is reabsorbed into the blood circulation system through the hepatointestinal circulation. Intestinal flora beta-glucuronidase plays an important role in the glucuronidation process of endogenous substances and drugs, and is highly related to gastrointestinal toxicity of drugs.

[0003] Irinotecan is a clinical treatment drug for diseases such as metastatic colorectal cancer, gastric cancer, non-small cell lung cancer, etc. After injection, irinotecan is first hydrolyzed by liver carboxylase to release the active metabolite SN-38; then, SN-38 is converted into glucuronidation product SN38G by UDP-glucuronosyltransferase, and is inactivated and enters the gastrointestinal tract through bile. Under the action of intestinal flora beta-glucuronidase, SN-38G is hydrolyzed to generate SN-38 again, and the strong cytotoxicity of SN-38 causes damage to intestinal epithelial cells, and then causes diarrhea, which limits the clinical application of irinotecan. Therefore, intestinal flora beta-glucuronidase is considered to be one of the important targets for treating drug-induced diarrhea induced by irinotecan. In addition, intestinal flora beta-glucuronidase is also directly related to gastrointestinal toxicity caused by non-steroidal anti-inflammatory drugs such as ketoprofen and diclofenac.

[0004] 7-hydroxy-diorcinol is a diphenyl ether compound, a colorless solid, with a molecular formula of C 14 H 14 O4 and a molecular weight of 246. The compound is easily soluble in methanol, and can be isolated from a fungus Aspergillus versicolor ZJUTE2, and has a chemical structure as shown in formula (I):

[0005]

[0006] At present, there is no relevant literature reported on whether the above-mentioned compound has beta-glucuronidase inhibitory activity, that is, whether the compound can be developed into a beta-glucuronidase inhibitor, which is still a research blank in the field. (III)SUMMARY

[0007] The application aims to solve the problems of the prior art, and provides application of a diphenyl ether compound isolated from Aspergillus versicolor ZJUTE2 in preparation of a β-glucuronidase inhibitor, so as to provide a lead structure for research and development of a new β-glucuronidase inhibitor.

[0008] To achieve the above application purposes, the application provides the following technical solutions.

[0009] The application provides application of a diphenyl ether compound in preparation of a β-glucuronidase inhibitor, wherein the diphenyl ether compound is 7-hydroxy-diplopygeol shown in formula (I), and the molecular formula is C 14 H 14 O4;

[0010]

[0011] Preferably, the β-glucuronidase is of an Escherichia coli origin.

[0012] The application further provides application of the diphenyl ether compound in preparation of a drug for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

[0013] The 7-hydroxy-diplopygeol is prepared according to the following method.

[0014] (1) Aspergillus versicolor ZJUTE2 is inoculated into a rice solid culture medium, and dark culture is carried out at 20-30 DEG C for 15-30 days to obtain a rice fermentation product; the Aspergillus versicolor ZJUTE2 is preserved in the China Center for Type Culture Collection, and the preservation date is January 13, 2022, the preservation number is CCTCC NO: M2022064, and the address is China, Wuhan, Wuhan University, which has been disclosed in patent application CN114806888A; the rice solid culture medium is a mixture of rice and distilled water, and the amount of distilled water is 1-5 mL / g (preferably 1.35 mL / g) based on the mass of rice;

[0015] (2) The rice fermentation product (preferably crushed) is added with an organic solvent, and room temperature (25-30 DEG C) extraction is carried out, the extract is concentrated to no liquid flow, and a crude extract is obtained;

[0016] (3) The crude extract of step (2) is suspended with water, extracted with an organic solvent, the organic phase is collected, and concentrated to dryness under reduced pressure to obtain an extract extract;

[0017] (4) The extract from step (3) was dissolved in methanol and subjected to MCI CHP20P column chromatography. Gradient elution was performed using a methanol / water mixture with a volume ratio of 30-100:70-0 as the eluent. Each gradient elution consisted of 2-5 column volumes (preferably 2) at a flow rate of 10-20 mL / min (preferably 15 mL / min). The methanol-water elution fraction with a volume ratio of 45:55-55:45 was collected and concentrated to dryness under reduced pressure to obtain a concentrate. The concentrate was dissolved in methanol, recrystallized at room temperature, and filtered to obtain 7-hydroxy-dimeric mossinol as shown in formula (I).

[0018] Preferably, before inoculating the *Aspergillus flavus* ZJUTE2 in step (1) into the rice solid culture medium, it is first activated, and then the activated bacterial suspension is inoculated into the rice solid culture medium at a volume concentration of 0.1-1% (preferably 1%). Activation refers to inoculating *Aspergillus flavus* ZJUTE2 into potato dextrose agar (PDA) medium and culturing it at 28°C for 7 days to activate the strain. The activated strain is then resuspended in sterile water containing 2% Tween 80 to obtain a bacterial suspension. The bacterial concentration in the bacterial suspension is 1×10⁻⁶. 5 -1×10 8 Cells / mL, preferably 1×10 6 The PDA culture medium consists of 200 g / L potato, 20 g / L glucose, and 18 g / L agar, with distilled water as the solvent and natural pH.

[0019] Preferably, the fermentation culture conditions in step (1) are: dark culture at 28℃ for 20 days.

[0020] Preferably, the organic solvent in step (2) is 95% ethanol, methanol or acetone; the volume of the organic solvent used is 2-8 mL / g (preferably 5-6 mL / g) based on the dry weight of rice in the rice solid culture medium; the extraction is performed at least 3 times, each extraction time is 3-5 days, and the volume of the organic solvent used in each extraction is 4 mL / g based on the dry weight of rice in the rice solid culture medium.

[0021] Preferably, the volume of water used in step (3) is 2-5 mL / g (preferably 3.1 mL / g) based on the weight of the crude extract; the organic solvent is ethyl acetate, and the volume ratio of the organic solvent to water is 1:0.5-3; the extraction is performed 3-5 times, and the volume ratio of the organic solvent to water used in each extraction is preferably 1:1.

[0022] Preferably, the method of step (4) is: 1) the extract is dissolved in methanol and subjected to MCI CHP20P column chromatography, and gradient elution is carried out with methanol / water mixed solvents with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 as eluents, 2-5 (preferably 2) column volumes for each gradient elution, and a flow rate of 10-20 mL / min (preferably 15 mL / min); the methanol-water elution part with a volume ratio of 45:55-55:45 is collected, concentrated to dryness under reduced pressure, and a concentrate is obtained; 2) the concentrate of step 1) is dissolved in methanol and subjected to recrystallization, and filtration is carried out to obtain the compound represented by (I).

[0023] The diphenyl ether compound obtained from the fungus Aspergillus versicolor ZJUTE2 through simple extraction and separation has good β-glucuronidase inhibitory activity and is comparable to the positive drug D-glucaric acid 1,4-lactone (DSL).

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] (1) The diphenyl ether compound obtained from the fungus Aspergillus versicolor ZJUTE2 through simple extraction and separation by organic solvent extraction and column chromatography has the advantages of simple preparation method, rapidness, high purity of the obtained compound, culture time of 20 days, yield of 260 mg / Kg of rice, and purity of more than 95%;

[0026] (2) The diphenyl ether compound obtained by the present application has good β-glucuronidase inhibitory activity, and the IC 50 value is 56.59±1.79 μM, which is comparable to the positive drug D-glucaric acid 1,4-lactone, and is expected to be used for the treatment of drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs. In addition, the development of structure optimization based on this type of compound has important significance for the research and development of new drug-induced diarrhea treatment drugs. (IV) DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the H-NMR spectrum of compound 1. 1

[0028] Figure 2 is the C-NMR spectrum of compound 1. 13

[0029] Figure 3 is the high performance liquid chromatogram of compound 1.

[0030] Figure 4 ​​This is the concentration-dependent curve of compound 1 on β-glucuronidase.

[0031] Figure 5 This is the concentration-dependent curve of D-glucosidate 1,4-lactone on β-glucuronidase.

[0032] Figure 6 This is a Lineweaver Burk double reciprocal plot used to determine the type of inhibition of compound 1. (V) Detailed Implementation

[0033] The present invention will be further described in detail with reference to the accompanying drawings and embodiments. However, it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0034] The room temperature mentioned in this invention refers to 25-30℃. The PDA culture medium consists of: 200g / L potato, 20g / L glucose, and 18g / L agar, with distilled water as the solvent and natural pH.

[0035] Example 1: Fermentation culture of Aspergillus versicolor ZJUTE2, a fungus that colonizes Cordyceps sinensis.

[0036] 1. Activation of the strain

[0037] Take out the cryovial of Aspergillus versicolor ZJUTE2 strain stored at -80℃, thaw it at 4℃, and use a sterile cotton swab to take an appropriate amount of spore suspension from the cryovial in a clean bench, inoculate it into PDA medium, and incubate it in a constant temperature incubator at 28℃ for 7 days to activate the strain.

[0038] 2. Fermentation by bacterial strain

[0039] After 7 days of activation, the spores were washed off with 10 mL of sterile water containing 2% Tween 80 (v / v) and placed in 100 mL of sterile water containing 2% Tween 80 (v / v) to obtain a spore suspension (spore concentration of 1×10⁻⁶). 6 (spores / mL); 1 mL of spore suspension was transferred to 50 1 L Erlenmeyer flasks containing rice culture medium (100 g rice, 135 mL distilled water, sterilized at 121 °C for 20 min and then cooled), and incubated at 28 °C in the dark for 20 days to obtain 50 bottles of rice fermentation products.

[0040] Example 2 Preparation of Compound 1

[0041] (1) The 50 bottles of rice fermentation product prepared in Example 1 were mixed and mashed, 20 liters of 95% ethanol was added, and extraction was carried out at room temperature for 4 days. The filtrate was repeated 3 times (3 liters of 95% ethanol was added each time, and each extraction was carried out for 4 days). The extract was combined and concentrated to dryness under reduced pressure to obtain 475 g of a crude extract.

[0042] (2) The crude extract (475 g) obtained in step (1) was suspended in 1.5 L of distilled water, and extracted with 1.5 L of ethyl acetate for 3 times. The ethyl acetate phase was combined and concentrated to dryness under reduced pressure to obtain 45 g of an ethyl acetate extract.

[0043] (3) The 45 g of the ethyl acetate extract obtained in step (2) was dissolved in 100 mL of methanol and subjected to MCI CHP20P column chromatography (diameter d = 4 cm, height h = 60 cm). Gradient elution was carried out with methanol / water mixed solvents with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, respectively, at a flow rate of 15 mL / min, and 1.5 L (2 column volumes) was used for each gradient elution. The elution fraction of methanol / water = 45:55 to 55:45 (v / v) was collected, combined, and concentrated to dryness under reduced pressure to obtain 2.1 g of a concentrate.

[0044] (4) The 2.1 g of the concentrate obtained in step (3) was dissolved in 10 mL of methanol and recrystallized at room temperature. Filtration was carried out to obtain 1.3 g of compound 1.

[0045] Example 3 Structure identification and purity detection of compound 1

[0046] 1. Structure identification of compound 1

[0047] Compound 1: colorless solid. The molecular formula is C 14 H 14 O4; its 1 H-NMR( Figure 1 ) and 13 C-NMR( Figure 2 ) data are as follows: 1 HNMR(600MHz,CD3OD):δ H 6.55(1H,m,H-6),6.47(1H,m,H-2),6.38(1H,s,H-6’),6.31(1H,t,J=2.2Hz,H-4),6.30(1H,s,H-4’),6.23(1H,t,J=2.1Hz,H-2’),4.50(2H,s,H-7),2.24(3H,s,H-7’)。 13 C NMR(150MHz,CD3OD):δ C159.9 (C-5), 159.8 (C-3), 159.6 (C-l'), 159.5 (C-3'), 145.6 (C-l), 141.7 (C-5'), 112.1 (C-6'), 118.9 (C-4'), 109.5 (C-6), 109.2 (C-2), 105.7 (C-4), 104.4 (C-2'), 64.9 (C-7), 21.5 (C-7'). The above spectral data are consistent with those of 7-hydroxy-diorcinol reported in the literature (He P, Tian S S, Xu Y, et al. Three new phenyl ether derivatives from Aspergillus carneus HQ889708. Helv. Chim. Acta, 2015, 98, 819-822.).

[0048] 2. Purity test of compound 1

[0049] (1) Compound 1 prepared in Example 2 was dissolved in methanol and diluted quantitatively to prepare a sample solution having a concentration of 200 μg / mL.

[0050] (2) The sample solution was subjected to high performance liquid chromatography analysis under the following chromatographic conditions. Instrument: Agilent 1200 series; column: Cosmosil 5C18-PAQ (4.6 mm I.D. x 250 mm); mobile phase: methanol-water (35:65 - 80:20, v / v); flow rate: 1 mL / min; detection wavelength: 254 nm; injection volume: 50 μL; column temperature: room temperature. The chromatogram was recorded.

[0051] (3) The high performance liquid chromatography detection result of the sample to be tested is shown in Figure 3 . The retention time of compound 1 was 16.523 min, and the peak area normalization analysis showed that the purity was 96.5%.

[0052] Example 4 Evaluation of in vitro β-glucuronidase inhibitory activity of compound 1

[0053] 1. Screening of EcGUS inhibitors (final concentration of inhibitors: 100 μM)

[0054] (1) Reagents

[0055] Inhibitors: Compound 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution, which was used as needed.

[0056] Substrate: 4-nitrophenyl-β-D-glucopyranoside (PNPG, purchased from Sigma-Aldrich) was dissolved in PBS buffer to prepare a 2.5 mM solution, which was used immediately.

[0057] Reaction enzyme solution: β-glucuronidase (EcGUS) lyophilized powder (purchased from Sigma, enzyme activity 4724.48 kU / g) was dissolved in PBS buffer and diluted to 2.5 μg / mL as a reaction enzyme solution.

[0058] Positive control (DSL): D-saccharic acid 1,4-lactone (DSL, purchased from Sigma-Aldrich) was dissolved in DMSO to prepare a 10 mM solution as a positive control.

[0059] (2) Reaction: The reaction was carried out in a 96-well plate, which was divided into a blank group, an experimental group and a positive control group.

[0060] Blank group: reaction enzyme solution 10 μL + PBS 79 μL + 10% (v / v) DMSO aqueous solution 1 μL + substrate 10 μL;

[0061] Experimental group: reaction enzyme solution 10 μL + PBS 79 μL + inhibitor 1 μL + substrate 10 μL;

[0062] Positive control group: reaction enzyme solution 10 μL + PBS 79 μL + 10 mM positive control 1 μL + substrate 10 μL.

[0063] Each group was prepared in triplicate, and the reaction enzyme solution, PBS, inhibitor / positive control and substrate were added in order. The OD values at 0 min and 30 min (incubated at 37°C) were measured at 405 nm using an enzyme marker. The calculation showed that the inhibition rate of compound 1 on EcGUS was 71.63% at a final concentration of 100 μM, which was higher than that of the positive control DSL (inhibition rate 68.85%).

[0064] The specific calculation process is as follows:

[0065] ΔOD = OD 30min - OD 0min ;

[0066] ΔC PNP = ΔOD / 0.003262 (0.003262 is the correlation coefficient between absorbance and PNP solubility in the present application);

[0067] Relative activity (%) = experimental group ΔC PNP / blank group ΔC PNP ;

[0068] Inhibition rate (%) = 1 - relative activity (%).

[0069] 2、IC 50 Determination of values: the IC 50 values of compound 1 were determined, a series of inhibitor concentration points (such as 0.1, 1, 10, 20, 30, 50, 70, 100 μM) were set within the final concentration of 0.1-100 μM, and the reaction was carried out in a 96-well plate, with blank group, experimental group and positive control group.

[0070] Blank group: reaction enzyme solution 10 μL + PBS 79 μL + volume fraction 10% DMSO 1 μL + substrate 10 μL;

[0071] Experimental group: reaction enzyme solution 10 μL + PBS 79 μL + different concentrations of inhibitors 1 μL + substrate 10 μL;

[0072] Positive control group: reaction enzyme solution 10 μL + PBS 79 μL + positive control 1 μL + substrate 10 μL.

[0073] Each group was set with 3 parallels, and the reaction enzyme solution, PBS, inhibitor / positive control, and substrate were added in order, and then the OD values at 0 min and 30 min were measured at 405 nm wavelength on the enzyme marker, respectively (incubated at 37°C during the period), the relative activity values of the inhibitor under different concentrations of EcGUS were calculated, and finally the lg value was obtained by taking the derivative of the inhibitor concentration (μM) with 10 as the base, and taking the lg value as the abscissa and the relative activity as the ordinate, the IC 50 curve graph Figure 4 , Figure 5 ) was drawn by using Graphad Prism 8.0 software, and the IC 50 value of the inhibitor / positive control on EcGUS was analyzed by the software, the IC 50 value of compound 1 on EcGUS was 56.59±1.79 μM, which was equivalent to the positive control DSL (IC 50 = 53.70±0.85 μM).

[0074] 3, Study on the inhibition type of compound 1 on EcGUS: Compound 1 was prepared into a solution with a concentration of 0.3, 0.5, 0.7 mM (i.e. a final concentration of 30, 50, 70 μM) with PBS, and the substrate was prepared into a solution with a concentration of 2, 3, 5, 10 mM (i.e. a final concentration of 200, 300, 500, 1000 μM) with PBS. The arrangement and combination table of different concentrations of pNPG and compound is shown in Table 1.

[0075] Table 1 Arrangement and combination table of different concentrations of pNPG and compound

[0076]

[0077] Note: C PNPG C represents the final concentration of the substrate. IN The final concentration of compound 1 is indicated by О, which represents a sample well in a 96-well plate. Each concentration combination is performed in triplicate.

[0078] The reaction was carried out in a 96-well plate. The reaction system was as follows: 10 μL of enzyme solution + 79 μL of PBS + 1 μL of different concentrations of inhibitor + 10 μL of different concentrations of substrate. Each combination was set up in triplicate. The enzyme solution, PBS, inhibitor, and substrate were added in the order of enzyme solution, PBS, inhibitor, and substrate. The absorbance was measured at 405 nm wavelength at 0 min and 30 min (incubated at 37 °C during the period) using a microplate reader. The PNP concentration difference corresponding to different concentration combinations was calculated according to step 1. Finally, 1 / V (μmol / min / mg) and 1 / PNPG values ​​were calculated. V (μmol / min / mg) is the catalytic rate of the enzyme, which represents the molar amount of product produced per milligram of enzyme per minute under certain conditions of temperature, pH, and substrate concentration.

[0079] The calculation process is as follows:

[0080] 1 / V(μmol / min / mg)=1 / (ΔC PNP *100 / 10 / 30 / 1); 1 / PNPG=1 / ΔC PNP ;

[0081] Where, ΔC PNP The values ​​represent the concentration difference of PNP in the system at 0 min and 30 min, 100 represents a reaction system of 100 μL, 10 represents the amount of enzyme added of 10 μL, 30 represents a reaction time of 30 min, and 1 represents an enzyme concentration of 1 μg / mL.

[0082] Finally, the suppression curve of the double reciprocal was plotted using Graphad Prism 8.0 software. Figure 6 The type of inhibitor is determined based on the intersection of the curves. Figure 5 The images show an intersection in the third quadrant, indicating that compound 1 inhibits β-glucuronidase in a mixed manner, binding to both the active and allosteric sites of the enzyme.

[0083] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. The use of a diphenyl ether compound of formula (I) in the preparation of a β-glucuronidase inhibitor, characterized in that, The inhibitor is a drug for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs; the β-glucuronidase is derived from Escherichia coli; Formula (I).

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