Use of a diaryl ether-pyrazole heterocyclic compound in the treatment of microbial infections

Abstract

The application belongs to the field of pharmaceutical chemistry, and discloses a new use of a diaryl ether-pyrazole heterocyclic compound in preventing and treating agricultural pathogenic fungal diseases caused by Botrytis cinerea Pers., Fusarium graminearum Schw., Rhizoctonia solani, Phytophthora capsici Leonian., Sclerotinia sclerotiorum, and in preventing and treating plant diseases caused by Xanthomonas oryzae ACCC 11602, Xanthomonas axonopodis pv.Citri, Pseudomonas soli macerae and Erwinia aroideae, and diseases caused by human pathogenic bacteria Escherichia coli ATCC 25922, Staphyloccocus aureus sp.Newman and Candida albicans ATCC 24433. The structural general formula of the diaryl ether-pyrazole heterocyclic compound is as follows: the diaryl ether-pyrazole heterocyclic compound has the characteristics of simple structure, easy synthesis, low price and wide use, and through activity determination, it is found that the diaryl ether-pyrazole heterocyclic compound has clear structure-activity relationship, a wide bactericidal spectrum and further research and development value.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and discloses a novel application of a diaryl ether-pyrazole heterocyclic compound in the control of agricultural pathogenic fungal diseases caused by *Botrytis cinerea* Pers., *Fusarium graminearum* Sehw., *Rhizoctonia solani*, *Phytophthora capsici* Leonian., *Sclerotinia sclerotiorum*, and *Fusarium oxysporum*. It also discloses its application in the control of plant diseases caused by *Xanthomonas oryzae* ACCC11602 (the pathogen of rice bacterial blight), *Xanthomonas axonopodis* pv. Citri (the pathogen of citrus canker), *Pseudomonas sollamacearum* (the pathogen of tomato bacterial wilt), and *Erwinia aroideae* (the pathogen of soft rot), as well as the human pathogen *Escherichia coli* ATCC. Use in diseases caused by Staphylococcus aureus (25922) and Candida albicans (ATCC 24433). Background Technology

[0002] Microbial infections, caused by tiny microorganisms such as bacteria and fungi, are one of the main factors contributing to plant and animal diseases. Statistics show that over 1.5 million people die from fungal infections each year, and more than 1 billion are affected by them; Salmonella infections alone account for as many as 3.4 million cases annually, with over 2 million deaths. Clearly, fungal and bacterial diseases have severely impacted human quality of life, becoming one of the greatest global public health threats.

[0003] Currently, chemical control remains one of the main measures for controlling fungal and bacterial infections. However, with the long-term and extensive use of existing antimicrobial agents, antimicrobial resistance is increasing year by year, causing significant crop yield reductions and resulting in incalculable economic losses and casualties. Antimicrobial resistance has become a highly challenging global problem. Therefore, there is an urgent need to develop novel fungicides with entirely new chemical structures and mechanisms of action.

[0004] Building upon our previous screening and optimization of lead compounds, we further derivatized the 1,3-dicarbonyl fragment using a diaryl ether-fluorinated 1,3-dicarbonyl compound as the parent core and employing a nitrogen heterocyclization strategy. We then tested the antibacterial activity of the target compounds against plant pathogenic fungi and bacteria, as well as human pathogenic fungi and bacteria. The results showed that the diaryl ether-pyrazole heterocyclic compound exhibited significant antibacterial activity against some of the aforementioned pathogens. These activity results demonstrate that compounds with diaryl ether-pyrazole heterocyclic compounds as structural units possess novel structures, broad-spectrum activity, and high efficacy, making them worthy of further research and development as antibacterial agents. Summary of the Invention

[0005] The purpose of this invention is to provide the use of diaryl ether-pyrazole heterocyclic compounds in the prevention and control of plant pathogenic fungi and human pathogenic fungi, specifically for the prevention and control of plant diseases caused by *Rhizoctonia solani*, *Citrus canker*, *Rhizoctonia solani*, *Soft rot*, *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, *Fusarium graminearum*, *Botrytis cinerea*, *Fusarium oxysporum*, and *Phytophthora capsici*, as well as human infections caused by *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*.

[0006] To achieve the above objectives, the present invention provides the following technical method:

[0007] The application of a diaryl ether-pyrazole heterocyclic compound in the resistance to plant pathogenic fungi and human pathogenic fungi infections. The general structural formula I of the diaryl ether-pyrazole heterocyclic compound is as follows:

[0008]

[0009] Among them, plant bacteria include *Xanthomonas oryzae* ACCC 11602, the pathogen of rice bacterial blight, *Xanthomonas axonopodis* pv. Citri, the pathogen of tomato bacterial wilt, and *Erwinia aroideae*; plant fungi include *Botrytiscinerea* Pers., *Fusarium graminearum* Sehw., *Rhizoctonia solani*, *Phytophthora capsici* Leonian., *Sclerotinia asclerotiorum*, and *Fusarium oxysporum*; and human pathogens include *Escherichia coli* ATCC 25922 and *Staphylococcus aureus*. sp. Newman) and Candida albicans (Candida albicans ATCC 24433).

[0010] The diaryl ether-pyrazole heterocyclic compound provided by this invention, as a novel bactericide, has the following advantages:

[0011] 1) Diaryl ether-pyrazole heterocyclic compounds have excellent inhibitory effects on plant pathogens and human pathogens, and are characterized by simple structure, cheap raw materials and easy synthesis.

[0012] 2) Diaryl ether-pyrazole heterocyclic compounds exhibit high bactericidal activity. Some plant fungi show inhibition rates exceeding 50% even at drug concentrations below 3.00 μg / mL.

[0013] 3) Diaryl ether-pyrazole heterocyclic compounds exhibit specific activity against Rhizoctonia solani. Most diaryl ether-pyrazole heterocyclic compounds show significant antifungal activity against the agricultural fungus Rhizoctonia solani and can be further designed and developed as lead compounds for the search for effective drugs against Rhizoctonia solani. Detailed Implementation

[0014] To better understand the present invention, the following specific embodiments further illustrate the above-described content of the present invention. However, this should not be construed as a limitation of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0015] Example 1: Synthesis of diaryl ether-pyrazole heterocyclic compounds:

[0016]

[0017] The synthesis method of the compound described in this invention is carried out according to the following reaction formula:

[0018] (1) The preparation methods of the compounds involved in general formula I are as follows:

[0019] Experimental section:

[0020] A diaryl ether-substituted fluorinated 1,3-dicarbonyl compound (3.33 mmol) was dissolved in AcOH (7 mL), and hydrazine hydrate (0.41 mL, 13.32 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 3 hours, and H₂O was slowly added until no solid precipitate formed. The solid was collected by filtration and washed with H₂O and n-hexane. The solid was purified by column chromatography to give a 1H-pyrazole derivative.

[0021] The structural formula and structural characterization of the compound described in this patent are shown in the table below:

[0022] Table 1. Structural formulas and structural characterizations of the compounds described in this patent.

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] Example 2: Determination of the anti-agricultural pathogenic fungal activity of diarylene-pyrazole heterocyclic compounds

[0031] The agricultural pathogens used in this experiment were strains preserved at 4℃ in the laboratory, and the culture medium used was potato agar-dextrose medium (PDA). PDA medium formula: 200g potato (peeled), 20g glucose, 15g agar, 1000mL distilled water, natural pH.

[0032] PDA culture medium preparation method: Wash and peel potatoes, weigh 200g and cut into small pieces. Boil in distilled water for about 20 minutes (until the potato pieces are soft but not mushy). Filter through eight layers of gauze, add distilled water to make up to 1000mL, add 15g agar and 20g glucose, stir to dissolve completely, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and cool before use. Indoor activity determination uses the mycelial growth rate method.

[0033] Strain activation: Incubate agricultural pathogens on PDA plates at 25°C for 3–6 days.

[0034] Preparation of drug-coated plates: Heat and melt PDA culture medium, cool to 45-50℃, and add different concentrations of mixtures to prepare drug-coated plates.

[0035] Inoculation and cultivation: In a clean bench, use a punch to make a 5mm diameter mycelium cake at the edge of the mycelium after 3-6 days of cultivation (with the growth conditions as uniform as possible), then use an inoculation needle to pick it up and place it in the center of the plate. Finally, incubate it upside down in an incubator (25℃).

[0036] Results determination: After the hyphae in the blank control group were fully grown, the growth diameter of the hyphae in the drug-treated group was measured by the cross-cross method, and the inhibition rate was calculated.

[0037] Inhibition rate (%) = (control mycelium diameter - treated mycelium diameter) / (control mycelium diameter - mycelium cake diameter) × 100. Three parallel experiments were set up for each concentration. The inhibition rate of the compounds was measured and is shown in Table 2.

[0038] Table 2. Antifungal effect of diaryl ether-pyrazole heterocyclic compounds against agricultural pathogenic fungi (concentration 50 μg / mL)

[0039]

[0040] Note: "-" indicates that no antibacterial activity was detected at this concentration; " / " indicates that the strain was not measured.

[0041] Table 2 (continued). Antifungal effect of diaryl ether-pyrazole heterocyclic compounds against agricultural pathogenic fungi (concentration 50 μg / mL)

[0042]

[0043] "-" indicates that no antibacterial activity was detected at this concentration, and " / " indicates that the antibacterial activity of this bacterial species was not tested.

[0044] The above results indicate that the diaryl ether-pyrazole heterocyclic compound involved in this invention exhibits excellent antibacterial effects against some pathogens. By reducing the concentration, the half-maximal effective concentration (EC50) of the highly active compound was determined using SPSS software. 50 The activity data obtained are shown in Table 3.

[0045] Table 3. EC5 activity of highly active diaryl ether-pyrazole heterocyclic compounds against agricultural pathogenic fungi 50 value

[0046]

[0047] Note: "-" indicates that no antibacterial activity was detected at this concentration; " / " indicates that the strain was not measured.

[0048] As shown in Tables 2 and 3, the bioassay results indicate that the diaryl ether-pyrazole heterocyclic compounds involved in this invention possess certain antibacterial activity against the five plant pathogenic fungi tested. In particular, all the diaryl ether-pyrazole heterocyclic compounds exhibited significant antibacterial activity against *Rhizoctonia solani*, with compound BZ-11 showing the best antibacterial activity against *Rhizoctonia solani*, and its EC50... 50 The value is 3.000 μg / mL. In summary, the diaryl ether-pyrazole heterocyclic compound described in this invention has value for further research and development in agriculture.

[0049] Example 3: Determination of the anti-plant pathogenic bacteria, human pathogenic fungi, and bacterial activity of diarylene-pyrazole heterocyclic compounds.

[0050] The bacterial strains used in this experiment were cryopreserved in the laboratory at -80℃ with 30% glycerol. The cryopreserved strains were removed and streaked onto NB solid medium for plant bacteria (beef extract: 3g, peptone: 5g, yeast extract: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH 7.0; sterilized at 121℃ for 20min), and incubated at 28℃ (37℃) until single colonies appeared. Single colonies from the solid medium were then transferred to NB liquid medium for plant bacteria (beef extract: 3g, peptone: 5g, yeast extract: 1g, sucrose: 10g, distilled water: 1L; sterilized at 121℃ for 20min) and incubated at 28℃ (37℃) with shaking at 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted with the appropriate liquid medium to approximately 10... 6 CFU / mL is prepared for use. Dissolve the compounds separately in DMSO, add them to the liquid culture medium, mix thoroughly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of approximately 10... 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, resulting in a final drug concentration of 100 μg / mL. A control of 100 μL of bacterial culture containing an equal amount of DMSO was used. The 96-well plates were incubated at 28℃ (37℃) for 24-48 h until bacterial growth was observed in the control group. The OD value (OD) of the bacterial culture in each well was measured using a microplate reader. 600In addition, the OD values ​​of 100 μL of liquid culture medium and a 100 μg / mL drug concentration were measured to correct for the OD values ​​caused by the culture medium and the drug itself. The formulas for calculating the corrected OD value and inhibition rate are as follows:

[0051] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0052] Inhibition rate = (OD value of bacterial suspension in the control medium after correction - OD value of bacterial suspension in the drug-containing medium after correction) / OD value of bacterial suspension in the control medium after correction × 100%

[0053] All experiments were conducted in triplicate. The active compound-containing liquid culture medium was diluted twofold in 96-well plates to obtain a series of 50 μL concentrations of the drug-containing medium. The inhibition rate corresponding to each concentration was then determined using the same experimental method as in Example 3. The method for determining the antibacterial activity of the 1,3-dicarbonyl compound against human fungi and bacteria was the same as for plant pathogenic bacteria, except that the culture medium was replaced with yeast extract glucose (YEPD) medium (for fungi) and hydrolyzed casein (MH) medium (for bacteria), respectively. The inhibition rates of the compounds are shown in Table 4, and the MIC values ​​are shown in Table 5.

[0054] Table 4. Antibacterial activity of diaryl ether-pyrazole heterocyclic compounds against plant pathogenic bacteria, human pathogenic fungi, and bacteria (100 μg / mL)

[0055]

[0056] Note: "-" indicates that no antibacterial activity was detected at this concentration; " / " indicates that the strain was not measured.

[0057] Continued from Table 4. Antibacterial activity of diarylene-pyrazole heterocyclic compounds against plant pathogenic bacteria, human pathogenic fungi, and bacteria (100 μg / mL)

[0058]

[0059] Note: "-" indicates that no antibacterial activity was detected at this concentration; " / " indicates that the strain was not tested.

[0060] Table 5. MICs of diaryl ether-pyrazole heterocyclic compounds against plant pathogenic bacteria, human pathogenic fungi, and bacteria. 90 value

[0061]

[0062] Note: "-" indicates that the antibacterial activity of the compound was not determined, and " / " indicates that the strain was not tested.

[0063] As shown in Tables 4 and 5, the bioassay results indicate that the diaryl ether-pyrazole heterocyclic compound of this invention exhibits excellent inhibitory effects on some of the tested strains. Specifically, BZ-13 showed an inhibition rate of over 90% against the test strain *Bacillus oryzae* at 100 μg / mL; compounds BZ-11 and BZ-13 showed an inhibition rate of over 90% against the test strain *Citrus canker* at 100 μg / mL; and compounds BZ-7, BZ-11, and BZ-13 showed an inhibition rate of over 90% against the test strain *Candida albicans* at 100 μg / mL. Further reduction of the dosage concentration revealed that diaryl ether-pyrazole heterocyclic compounds BZ-11 and BZ-13 still exhibited good antibacterial activity against *Citrus canker* at low concentrations, while most other diaryl ether-pyrazole heterocyclic compounds lost their antibacterial activity.

[0064] In summary, the diaryl ether-pyrazole heterocyclic compound of this invention exhibits excellent inhibitory activity against the plant pathogenic fungus Rhizoctonia solani. This diaryl ether-pyrazole heterocyclic compound can be further studied and developed as a lead compound for the search of effective drugs against Rhizoctonia solani.

Claims

1. The use of a diaryl ether-pyrazole heterocyclic compound BZ-1 to BZ-19 in the control of plant diseases caused by pathogenic bacteria and / or fungi, characterized in that, The structural formulas of the diaryl ether-pyrazole heterocyclic compounds BZ-1 to BZ-19 are respectively ； The diaryl ether-pyrazole heterocyclic compounds BZ-1 to BZ-6, BZ-8 to BZ-10, BZ-12, and BZ-14 to BZ-19 control citrus canker, a plant disease caused by pathogenic bacteria; the diaryl ether-pyrazole heterocyclic compounds BZ-7, BZ-11, and BZ-13 control one or both of rice bacterial blight and citrus canker. The diaryl ether-pyrazole heterocyclic compound BZ-1-BZ-19 controls plant diseases caused by pathogenic fungi, including one or more of the following: Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Phytophthora capsici.

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