Application of 2-fluorinated naphthol compounds

Abstract

The present invention discloses an application of 2-fluorinated naphthol compounds, which belongs to the field of bioengineering. Specifically, the application of 2-fluorinated naphthol compounds in the preparation of drugs for treating infectious diseases caused by fungi. The present invention proves through experiments that 2-fluorinated phenol compounds can effectively inhibit the growth of fungi, and the cytotoxicity thereof is lower than that of naphthol, and the 2-fluorinated phenol compounds have higher tolerance to salt ions, acid and alkali, temperature and oxidants, good stability, and have the potential to be developed into antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to the application of 2-fluorinated naphthol compounds. Background Art

[0002] Due to the strong electron-withdrawing properties of fluorine atoms and their relatively small atomic radius, fluorine-containing compounds usually have a variety of excellent physical and chemical properties and biological activities. In drug design, by introducing fluorine atoms or fluorine-containing groups into organic molecules, the binding affinity, metabolic stability, cell membrane permeability and other biological activities of certain drug molecules can be improved. Therefore, fluorine-containing compounds occupy an important position in the research and development of new drugs and are widely used in the fields of medicine, pesticides, functional materials, etc. Fluorinated phenolic compounds, such as fluorinated naphthol, are important intermediates for the synthesis of functional materials. They are special skeletons of drug molecules and exist in a variety of biologically active molecules. They have potential in the preparation of antibacterial drugs. Fluorinated phenolic compounds, such as fluorinated naphthol, are important intermediates for the synthesis of functional materials. They are special skeletons of drug molecules and exist in a variety of biologically active molecules. They have potential in the preparation of antibacterial drugs. 2-Fluorinated naphthol compounds are products prepared by catalytic ring expansion reaction of indanone compounds (Chem. Commun., 2015, 51, 15362.). Based on the known 2-fluorinated naphthol compounds, their medicinal value needs to be further explored and developed. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an application of a 2-fluorinated naphthol compound in the preparation of an antifungal drug, wherein the 2-fluorinated naphthol compound can effectively inhibit fungal growth.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: Use of a 2-fluorinated naphthol compound in the preparation of a drug for treating infectious diseases caused by fungi, wherein the molecular structure of the 2-fluorinated naphthol compound is shown in formula (I):

[0005]

[0006] Furthermore, the fungus is Candida albicans, Candida tropicalis, Candida parapsilosis, Fusarium graminearum or Aspergillus fumigatus.

[0007] Advantages and beneficial effects of the present invention: The present invention proves through experiments that 2-fluorinated phenol compounds can effectively inhibit the growth of fungi, have lower cytotoxicity than naphthol, have higher tolerance to salt ions, acids and bases, temperature and oxidants, have good stability, and have the potential to be developed into antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The 2-fluorinated naphthol compound (2d) of the present invention is1 H NMR spectrum;

[0009] Figure 2 The 2-fluorinated naphthol compound (2d) of the present invention is 13 C NMR spectrum;

[0010] Figure 3 The 2-fluorinated naphthol compound (2d) of the present invention is 19 F NMR spectrum;

[0011] Figure 4 The figure is a comparative measurement of the hemolytic activity of the 2-fluorinated naphthol compound (2d) of the present invention and naphthol;

[0012] Figure 5 The cytotoxicity comparison chart of the 2-fluorinated naphthol compound (2d) of the present invention and naphthol is shown in FIG.

[0013] (a) is the toxicity graph of 2d and 1-naphthol to human embryonic kidney cells (HEK 293T),

[0014] (b) is the toxicity graph of 2d and 1-naphthol to mouse mononuclear macrophages (RAW 264.7).

[0015] (c) is the toxicity graph of 2d and 1-naphthol to porcine intestinal epithelial cells (IPEC J2);

[0016] Figure 6 is a bactericidal kinetic curve of the 2-fluorinated naphthol compound (2d) of the present invention;

[0017] (a) is the bactericidal kinetic curve of killing Fusarium graminearum at 1×MFC concentration for 2 days.

[0018] (b) is the bactericidal kinetic curve of killing Aspergillus fumigatus at 1×MFC concentration for 2 days;

[0019] Figure 7 The figure is a comparative measurement chart of the respiratory chain inhibition of the 2-fluorinated naphthol compound (2d) of the present invention and naphthol, wherein:

[0020] (a) is a graph showing the level of respiratory chain dehydrogenase in Aspergillus fumigatus cells after compound treatment.

[0021] (b) is a graph showing the levels of respiratory chain dehydrogenases in Fusarium graminearum cells after treatment with compounds;

[0022] Figure 8 is a graph showing the measurement of membrane fluidity of the 2-fluorinated naphthol compound (2d) of the present invention, wherein:

[0023] (a) is the change of membrane fluidity of Aspergillus fumigatus cells after 2 days of treatment.

[0024] (b) is the change of cell membrane fluidity of Fusarium graminearum after 2 days of treatment;

[0025] Fig. 9 This is a measurement diagram of the active oxygen aggregation caused by the 2-fluorinated naphthol compound (2d) of the present invention and naphthol, wherein:

[0026] (a) is the graph of ROS levels in Aspergillus fumigatus cells induced by compounds.

[0027] (b) is a graph showing the level of ROS in Fusarium graminearum cells under the induction of compounds;

[0028] Fig.10 This is a scanning electron microscope image of the 2-fluorinated naphthol compound (2d) of the present invention inhibiting Fusarium graminearum, wherein:

[0029] (a) is the SEM image of Fusarium graminearum without 2d treatment.

[0030] (b) is the SEM image of Fusarium graminearum after 2 days of treatment. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0032] Example 1

[0033] The method for preparing 2-fluorinated naphthol compound (2d) by catalytic ring expansion reaction of indanone is as follows:

[0034] At room temperature, 3-oxo-indane-acetate 1d (0.5 mmol), tetrabutylammonium bromide (TBAB) (0.1 eq, 0.05 mmol), TMSCF 2 Br (1.5eq, 0.75mmol) and toluene (2.0mL). After the pressure tube was plugged with a polytetrafluoroethylene stopper and placed in an oil bath at 110°C for 3.0 hours, the system was cooled to room temperature and TMSCF was added to the reaction system. 2 Br (1.5eq, 0.75mmol), the sealed pressure tube was placed in an oil bath at 110°C and the reaction continued until the starting material was completely consumed as monitored by TLC. The reaction system was cooled to room temperature, tetrabutylammonium fluoride (TBAF) (THF solution, M = 1.0mol / L) (0.1eq, 0.1mmol) was added thereto, and stirring was continued at room temperature until the intermediate was completely converted as detected by TLC. After the reaction was complete, the reaction system was quenched with dilute hydrochloric acid (20mL) and quenched with CH 2 Cl 2 (3 × 15 mL) and extracted with anhydrous NaSO 4The organic phase was dried, and the organic solvent was then evaporated under vacuum. Finally, the crude product was subjected to column chromatography (developing solvent: petroleum ether, ethyl acetate) to obtain product 2d.

[0035]

[0036] According to compound 2d 1 H NMR, 13 C NMR, 19 The structure of the compound was determined by F NMR spectroscopy ( Figure 1 , Figure 2 and Figure 3 Specifically, the compound was purified by column chromatography (petroleum ether:ethyl acetate=10:1), white crystals (95.72 mg, 87%): mp 132-134°C.

[0037] 1H NMR (500MHz, CDCl 3 )δ2.37(s,1H),5.68(s,1H),7.17-7.24(m,2H),7.33(dd,J=5.0Hz,J=9.0Hz,1H),7.77(d,J=9.0Hz,2H),7.86(d,J=2.0Hz,2H). 13 C NMR (125 MHz, CDCl 3 )δ21.2,113.0(d,J=6.9Hz,1C),115.3(d,J=21.9Hz,1C),120.2(d,J=7.6Hz,1C),120.8,125.8( d,J=3.4Hz,1C),129.0,129.2,137.2(d,J=14.5Hz,1C),147.0(d,J=232.3Hz,1C),148.4,169.9. 19 F NMR (470 MHz, CDCl 3 )δ(-146.20)-(-146.16)(m,1F).HRMS(ESI-TOF)calcd forC 12 H 10 FO 3 + ([M+H] + )221.0608, found 221.0602.

[0038] Example 2: The prepared 2-fluorinated naphthol compound (2d) was tested for in vitro antibacterial activity, hemolytic activity, cytotoxicity, bactericidal power, stability, etc.:

[0039] 1. Determination of antibacterial activity: Prepare the compound into a certain storage solution for use. Use the microbroth dilution method to determine the minimum inhibitory concentration of the compound. Use 0.01% acetic acid (containing 0.2% BSA) as the diluent and use the two-fold dilution method to prepare a series of gradient compound solutions. Take 50 μL of the above solution and place it in a 96-well cell culture plate, then add equal volumes of the test bacterial solution (0.5-1×10 5 CFU / mL) in each well. Set up a positive control (containing bacterial solution but no compound) and a negative control (containing neither bacterial solution nor compound). Incubate at 30℃ for 24h. The minimum inhibitory concentration is the concentration when no turbidity is seen at the bottom of the well. The test results are shown in Table 1.

[0040] Table 1 Antibacterial activity of compounds (mg / ml)

[0041]

[0042]

[0043] It can be seen from Table 1 that the 2-fluorinated naphthol compound (2d) has a more efficient antibacterial activity against the tested fungi, indicating that the 2-fluorinated naphthol compound (2d) has the potential to become a new generation of antifungal drugs.

[0044] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, centrifuge at 1000g for 5 minutes, collect red blood cells; wash with PBS 3 times, and resuspend with 10 mL PBS; take 50 μL of red blood cell suspension and 50 μL of compound solution of different concentrations dissolved in PBS, mix them evenly, and incubate them in a 37°C incubator for 1 hour; take out after 1 hour, centrifuge at 4°C and 1000g for 5 minutes; take out the supernatant and measure the absorbance value at 570nm with an enzyme marker; take the average value of each group and compare and analyze. 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 was used as a positive control. The minimum hemolytic concentration is the concentration of the compound when the compound causes a hemolysis rate of 10%. See the test results. Figure 4 And Table 2.

[0045] Table 2 shows the determination of hemolytic activity of 2-fluorinated naphthol compound (2d)

[0046]

[0047] *When the minimum hemolytic concentration is >1 mg / ml, use 2 mg / ml to calculate the selectivity index

[0048] Comprehensive analysis of the antibacterial and hemolytic activities of the compounds can be used to more comprehensively evaluate the biological activity of the compounds through the selectivity index (ratio of hemolytic concentration to inhibitory concentration). As can be seen from Table 2, the 2-fluorinated naphthol compound (2d) has a good selectivity index relative to naphthol, indicating that the designed 2-fluorinated naphthol compound (2d) has the potential to become a new antibacterial drug.

[0049] 3. Cytotoxicity assay: The cells frozen in liquid nitrogen were revived and inoculated in a culture medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% CO. 2 The cultured cells were digested with 0.25% trypsin and the volume was adjusted to 2-4×10 5 50 μL of cell suspension was mixed with 50 μL of compounds of different concentrations in a 96-well plate and incubated at 37°C and 5% CO 2 Incubate for 24 hours under the same conditions, then add 25 μL MTT (5 mg / mL) to each well and continue incubation for 4 hours. After the incubation, discard the supernatant, dissolve the crystals at the bottom of the well with 100 μL DMSO, and measure the absorbance of each well at 570 nm using an enzyme reader. The culture medium wells were used as blank controls. The test results are shown in Figure 5 And Table 3.

[0050] Table 3 shows the determination of cytotoxicity of 2-fluorinated naphthol compound (2d)

[0051]

[0052] from Figure 5 As shown in Table 3, 2-fluorinated naphthol compounds can selectively kill fungi, indicating that they have lower cytotoxicity than naphthol and have the potential to become a new generation of antifungal drugs.

[0053] 4. Stability determination: Add different concentrations of salt ions to the 1640 compound dilution, and simulate the effects of different temperatures, oxidant action time, and pH conditions on the compound, and determine the changes in the MIC value of the drug against Aspergillus fumigatus according to the above antibacterial activity determination method. The final salt ion concentrations determined are: 150mM NaCl, 4.5mM KCl, 6μM NH 4 Cl, 8 μM ZnCl 2 , 1 mM MgCl 2 and 4 μM FeCl 3 The final temperature values ​​are: 40 degrees, 70 degrees, 100 degrees; the final reaction time with the oxidant is 4h, 8h, 12h; the pH is pH = 4, pH = 7, pH = 10. The test results are shown in Tables 4 and 5.

[0054] Table 4 shows the salt ion and temperature stability of 2-fluorinated naphthol compound (2d)

[0055]

[0056] Table 5 shows the oxidant and pH stability of 2-fluorinated naphthol compound (2d)

[0057]

[0058] It can be seen from Tables 4 and 5 that 2-fluorinated naphthol compounds still have good antibacterial activity in different salt ion concentrations, temperatures, oxidant action time, and acid-base environments, indicating that they have high tolerance to salt ions, acid-base, temperature and oxidants, good stability, and certain clinical application potential.

[0059] 5. Bactericidal kinetics determination:

[0060] (1) Preparation of bacterial solution: Fungi: Take the strain frozen at -20°C and streak it on the corresponding solid culture medium and culture it at 37°C overnight. Then pick a single colony and inoculate it in PDB, culture it at 220rpm and 37°C until the logarithmic growth phase, adjust its concentration to OD600 = 0.1 with PBS, and finally dilute it 1000 times with PBS to 0.5-1×10 5 CFU / mL.

[0061] (2) Determination of bactericidal kinetics curve: The bacterial solution was mixed with 1×MBC concentration compound, and 50 μL of samples were diluted at different time points (0, (10s) 15s, 30s, 45s, 60s, 3min, 5min, 10min, 15min, 30min, 60min), and spread on the corresponding solid culture medium for culture. The fungal survival rate at each time point was then calculated and the curve was drawn. The test results are shown in Figure 6 .

[0062] from Figure 6 It can be seen that at a concentration of 1×MFC, 2-fluorinated naphthol compounds killed 100% of Fusarium graminearum and Aspergillus fumigatus within 15 minutes, showing a faster bactericidal rate, indicating that 2-fluorinated naphthol compounds have strong bactericidal activity and can be widely used as a new antifungal drug in the future.

[0063] 6. Respiratory chain inhibition:

[0064] (1) Preparation of bacterial solution: Use Tris-HCl buffer to adjust the concentration of bacterial solution to 5×10 5 CFU / mL.

[0065] (2) Sample treatment: 1 mg / mL RT, 0.1 mol / L glucose solution and 0.05 mol / L Tris-HCl buffer were mixed in a ratio of 1:1:1. The mixed solution was then used to dilute the drug to different concentrations, and then the drug solutions of different concentrations were mixed with the bacterial solution in a 96-well plate. The mixed solution without concentration was set as the negative control. The 96-well plate was placed in a 37°C incubator and incubated for 12 h.

[0066] (3) Result determination: The absorbance value of the 96-well plate at a wavelength of 492 nm was determined using an ELISA instrument. Three parallels were set for each drug, and the test was repeated three times independently. The test results are shown in Figure 7 .

[0067] from Figure 7 It can be seen that the inhibitory effect of 2-fluorinated naphthol compounds on the respiratory chains of Fusarium graminearum and Aspergillus fumigatus is stronger than naphthol, indicating that 2-fluorinated naphthol compounds have a good inhibitory effect on the respiratory chain and can be widely used as a new antifungal drug.

[0068] 7. Membrane fluidity:

[0069] (1) Wash the collected cells after centrifugation three times with PBS solution, and adjust the bacterial solution concentration to OD600 = 0.4 with PBS (10 mM, pH = 7.4) solution. Then, add Laurdan with a final concentration of 20 μM to the bacterial solution, and incubate the mixture in a 37°C incubator in the dark for 30 minutes.

[0070] (2) Sample treatment: 50 μL of peptide solutions of different concentrations and 50 μL of the mixed solution were mixed evenly in a 96-well plate in the dark. The mixed solution without peptide was set as the negative control.

[0071] (3) Result determination: The fluorescence intensity of each well was measured using a multifunctional microplate reader under the conditions of an excitation wavelength of 350 nm and an emission wavelength of 430 nm-500 nm. Three parallels were set for each polypeptide, and the experiment was repeated three times independently. The fluidity of the cell membrane was calculated according to the following formula:

[0072] GP=(I440-I490) / (I440+I490)

[0073] GP is an indicator for evaluating changes in intracellular membrane fluidity; I440 nm is the fluorescence intensity of the polypeptide at an emission wavelength of 440 nm; and I490 nm is the fluorescence intensity of the polypeptide at an emission wavelength of 490 nm. Figure 8 .

[0074] Depend on Figure 8It can be seen that 2-fluorinated naphthol compounds can significantly increase the rigidity of the cell membrane of Fusarium graminearum and Aspergillus fumigatus, affect the homeostasis of the cell membrane, and make the cell membrane more easily ruptured.

[0075] 8. Reactive oxygen species (ROS) accumulation:

[0076] (1) Preparation of bacterial solution: The concentration of the conidia suspension was adjusted to 5×10 5 with hepes. Then, DCFH-DA was added to the bacterial solution at a final concentration of 200 μM, and the mixture was incubated in a 37°C incubator for 1 h 30 min.

[0077] (2) Sample processing: Mix the drug solutions of different concentrations and the bacterial solution in a 96-well plate. The mixture without peptide was set as negative control.

[0078] (3) Result determination: The fluorescence intensity of each well was measured using a multifunctional microplate reader under the conditions of an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Three parallels were set for each drug, and the experiment was repeated three times independently. The test results are shown in Fig. 9 .

[0079] Depend on Fig. 9 It can be seen that 2-fluorinated naphthol compounds can significantly cause the accumulation of reactive oxygen in Aspergillus fumigatus and Fusarium graminearum cells, resulting in cell apoptosis. Especially in Aspergillus fumigatus cells, the effect of 2-fluorinated naphthol compounds is much better than naphthol, indicating that the modified 2-fluorinated naphthol compounds can have antibacterial effects from more mechanisms.

[0080] 9. Scanning electron microscopy (SEM): After the Fusarium graminearum in the logarithmic phase was collected by centrifugation, it was resuspended in HEPES buffer to OD600 = 0.2, and then the polypeptide was added to the treated bacterial solution to make the final concentration 1×MFC, and the bacterial solution without the addition of polypeptide was used as a control. After incubation at 37°C for 1 hour, the fungal cells were collected and fixed overnight at 4°C by adding 600μL 2.5% glutaraldehyde. After fixation, the fungal cells were dehydrated with 50%, 70%, 90% and 100% ethanol in turn, and then replaced with 600μL tert-butanol-ethanol (1:1, v / v) and total tert-butanol for 15 minutes, followed by freeze-drying and coating. Finally, the samples were observed using a Hitachi S-4800 scanning electron microscope. The results are shown in Fig.10 .

[0081] Depend on Fig.10 It can be found that after being treated with 2-fluorinated naphthol compounds, the surface of F. graminearum ACCC 37687 showed obvious damage and wrinkles, and there was a tendency for the contents to leak out, indicating that 2-fluorinated naphthol compounds have a good bactericidal effect and can be widely used as a new antifungal drug in the future.

Claims

1. Use of a 2-fluorinated naphthol compound in the preparation of a drug for treating infectious diseases caused by fungi, wherein the molecular structure of the 2-fluorinated naphthol compound is as shown in formula (I): The fungus is Candida albicans, Candida tropicalis or Candida parapsilosis.

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