Application of amide compound or agrochemically acceptable salt thereof in preparation of antifungal drugs

Abstract

The invention belongs to the technical field of plant disease control, and particularly relates to application of amide compounds or agrochemically acceptable salts thereof in preparation of antifungal drugs. The amide compound or the acceptable salt of the amide compound shows remarkable broad-spectrum antibacterial activity on various plant pathogenic fungi such as rhizoctonia solani, tobacco target leaf spot bacteria and magnaporthe oryzae, and has no visible phytotoxicity symptom on plants in an effective concentration range. The action mechanism research shows that the amide compound or the acceptable salt thereof inhibits the growth of fungi through multiple ways: (1) the biological wall and cell membrane structures of the fungi are destroyed to cause the outflow of contents so as to play a bactericidal role; (2) the physiological function of fungal normal cells is interfered, so as to induce programmed death of fungal cells. The invention provides an important reference way for developing a high-efficiency novel agricultural bactericide.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease prevention and control, and more specifically, relates to the use of amide compounds or agrochemically acceptable salts thereof in the preparation of antifungal drugs. Background Art

[0002] Plant pathogenic fungi are among the most critical pests in global agricultural production. According to relevant statistics, fungal diseases cause approximately 25% annual crop yield losses, resulting in direct economic losses exceeding $200 billion. Traditional chemical fungicides, such as triazoles and benzimidazoles, are unable to meet the sustainable development needs of modern agriculture due to their limited target activity and the frequent emergence of resistance. For example, the mutation rate of Botrytis cinerea to benzimidazole fungicides has reached as high as 70%, significantly reducing the effectiveness of control. Furthermore, the activation of multidrug resistance efflux pumps further exacerbates pathogen resistance, increasing the difficulty of control.

[0003] Therefore, there is an urgent need to develop new and efficient antibacterial molecules to meet the challenges of current agricultural production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing antifungal drugs, such as drug resistance leading to poor efficacy, and to provide the use of amide compounds or agrochemically acceptable salts thereof in the preparation of antifungal drugs.

[0005] The present invention provides a method for preparing an antifungal drug using a protected amide compound or an agrochemically acceptable salt thereof, wherein the amide compound has the following structure:

[0006]

[0007] The present invention discovers for the first time that amide compounds of the aforementioned structure, or their agrochemically acceptable salts, exhibit significant broad-spectrum antifungal activity against a variety of plant pathogenic fungi, such as Rhizoctonia solani, tobacco leaf spot pathogen, and rice blast fungus, with no visible phytotoxicity to plants within the effective concentration range. Mechanism of action studies have shown that these amide compounds or their agrochemically acceptable salts inhibit fungal growth through multiple pathways: 1) disrupting the fungal biowall and cell membrane structure, leading to the outflow of fungal contents and exerting a fungicidal effect; and 2) interfering with the normal physiological functions of fungal cells, thereby inducing programmed cell death.

[0008] Furthermore, the agrochemically acceptable salt includes a hydrochloride. The salt form of Compound S1 releases Compound S1 to exert its activity in water or an aqueous environment, and thus the salt is merely a delivery form of Compound S1. While the solubility of Compound S1 is improved by preparing the salt form, the fungicidal effect still originates from the parent structure of Compound S1.

[0009] Furthermore, the hydrochloride of the amide compound has the following structure:

[0010] The hydrochlorides of the above-mentioned amide compounds can be purchased from the market or synthesized by oneself.

[0011] Furthermore, the synthesis method of the hydrochloride of the amide compound comprises the following steps:

[0012] N-boc-1,4-butanediamine was dissolved in CH2Cl2, 2-naphthoyl chloride and triethylamine were added under ice bath conditions, and the mixture was fully reacted at room temperature. The first post-treatment was performed to obtain a crude product. The crude product was purified and dissolved in a hydrochloric acid-ethyl acetate solution. The mixture was fully stirred and the second post-treatment was performed to obtain the hydrochloride of the amide compound.

[0013] Furthermore, the reaction time is 0.8 to 1.5 hours, preferably 1 hour.

[0014] Furthermore, the first post-treatment includes concentration and drying, specifically, the reaction product is concentrated by rotary evaporation and then vacuum dried to obtain a crude product.

[0015] Furthermore, the second post-treatment includes concentration and drying, specifically, concentrating the stirred product by rotary evaporation and then vacuum drying to obtain the hydrochloride of the amide compound.

[0016] Furthermore, the purification is to purify the obtained crude product by silica gel (200-300) column chromatography (dichloromethane / methanol=40:1).

[0017] Furthermore, the fungi include one or more of Rhizoctonia solani, Nicotiana tabacum target leaf spot pathogen, and Magnaporthe grisea.

[0018] Furthermore, the concentration of the amide compound or an agrochemically acceptable salt thereof is 0.1 to 300 mM, preferably 0.5 to 200 mM, and more preferably 2 to 200 mM.

[0019] Even more preferably, the concentration of the amide compound or the agrochemically acceptable salt thereof is 50-200 mM.

[0020] Furthermore, the amide compound or the agrochemically acceptable salt thereof has an antifungal effect by destroying the cell wall of the fungus.

[0021] Furthermore, the amide compound or agrochemically acceptable salt thereof exerts an antifungal effect by regulating the activity of antioxidant enzymes.

[0022] Furthermore, the antioxidant enzyme is catalase and / or superoxide dismutase.

[0023] Furthermore, the amide compound or an agrochemically acceptable salt thereof exerts an antifungal effect by inducing lipid peroxidation.

[0024] Furthermore, the antifungal drug includes an amide compound or an agrochemically acceptable salt thereof, and also includes an agrochemically acceptable carrier and / or excipient.

[0025] Furthermore, the dosage form of the antifungal drug is a solution, emulsion, suspension, powder, foam, paste, granule or aerosol.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The amide compounds or their acceptable salts disclosed herein exhibit significant broad-spectrum antifungal activity against a variety of plant pathogenic fungi, including Rhizoctonia solani, tobacco leaf spot pathogen, and rice blast fungus, with no visible phytotoxicity to plants within the effective concentration range. Mechanism of action studies have shown that these amide compounds or their agrochemically acceptable salts inhibit fungal growth through multiple pathways: 1) disrupting the fungal biowall and cell membrane structure, leading to the outflow of fungal contents and exerting a fungicidal effect; and 2) interfering with the normal physiological functions of fungal cells, thereby inducing programmed cell death. This invention provides an important reference for the development of highly effective, novel agricultural fungicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Graphs showing the in vitro protective effects of compound S1 (upper figure) and azoxystrobin (lower figure) at different concentrations against Rhizoctonia solani in Example 3.

[0029] Figure 2 Graphs showing the in vitro therapeutic effects of compound S1 (upper figure) and azoxystrobin (lower figure) at different concentrations on Rhizoctonia solani in Example 3.

[0030] Figure 3 1 is a bar graph showing the in vitro protective control effect (A) and therapeutic effect (B) of compound S1 and azoxystrobin at different concentrations against Rhizoctonia solani in Example 3.

[0031] Figure 4 Graphs showing the protective effects of different concentrations of compound S1 (upper figure) and azoxystrobin (lower figure) against Rhizoctonia solani in Example 4.

[0032] Figure 5 Graphs showing the in vivo therapeutic effects of compound S1 (upper figure) and azoxystrobin (lower figure) at different concentrations on Rhizoctonia solani in Example 4.

[0033] Figure 6Graph showing the effects of different concentrations of compound S1 (upper figure) and azoxystrobin (lower figure) on the sclerotium formation of Rhizoctonia solani in Example 5.

[0034] Figure 7 Graph showing the effects of different concentrations of compound S1 (upper figure) and azoxystrobin (lower figure) on the germination of Rhizoctonia solani sclerotia in Example 6.

[0035] Figure 8 This is a diagram showing the effect of compound S1 in Example 7 on the mycelial morphology of Rhizoctonia solani.

[0036] Figure 9 Effects of compound S1 on Rhizoctonia solani in Example 8: nucleic acid in hyphae cells (OD 260 ) and protein (OD 280 )’s release effect diagram.

[0037] Figure 10 This is a diagram showing the effects of compound S1 on four enzyme activities (A to D) of Rhizoctonia solani in Experimental Example 9. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0040] Example 1 Synthesis of Compound S1 Hydrochloride

[0041] The structure of compound S1 hydrochloride is as follows:

[0042]

[0043] Dissolve N-boc-1,4-butanediamine in 60 mL of dry CH2Cl2, add 2-naphthoyl chloride (5.0 mmol) dropwise at 0°C using a constant pressure dropping funnel, and then add triethylamine (12.5 mmol). After reacting at room temperature at 25°C for 1 hour, concentrate by rotary evaporation and dry in vacuo to obtain a crude product. The crude product is purified by silica gel (200-300) column chromatography (dichloromethane / methanol = 40:1). Then, add 10 mL of hydrochloric acid-ethyl acetate solution to the product obtained above and dissolve it. After stirring for 30 minutes, concentrate by rotary evaporation and dry in vacuo to obtain the product compound S1 hydrochloride. Its H NMR data are as follows:

[0044] 1H NMR (500MHz, DMSO-d6) δ8.71(t,J=5.7Hz,1H),8.46(s,1H),8.11–7.81(m,7H),7.55(tt,J=7. 1,5.2Hz,2H),3.36(d,J=5.9Hz,2H),2.83(q,J=6.4Hz,2H),1.65(dq,J=12.1,6.7Hz,4H).13C NMR (126MHz, DMSO-d6) δ171.54,139.28,137.35,137.09,133.90,132.84,132.64,132.63,132.49,131.65,129.41,43.87,43.72,31.37,29.71.

[0045] In subsequent testing, although the hydrochloride salt form of Compound S1 was used (to improve its solubility), the salt form of Compound S1 releases Compound S1 to exert its activity in water or an aqueous environment. Therefore, the salt is only the delivery form of Compound S1; its bactericidal activity still originates from the parent structure of Compound S1. For simplicity, "Compound S1" described in subsequent examples refers to its hydrochloride salt form.

[0046] Example 2 Inhibitory effect of compound S1 on Rhizoctonia solani, Magnaporthe oryzae, and tobacco leaf spot pathogen

[0047] The present invention systematically evaluated the in vitro antibacterial activity of compound S1 against Rhizoctonia solani, tobacco leaf spot pathogen, and rice blast fungus using a mycelial growth rate method. Because compound S1 ultimately exists as a hydrochloride with good solubility, distilled water was used to dissolve and prepare the test compound solution before the experiment. Within a clean bench, the test compound was thoroughly mixed with potato dextrose agar (PDA) and diluted to 10 mM. The solution was then evenly poured into 90 mm culture dishes. Azoxystrobin (SC) was used as a positive control, and an equal volume of distilled water was used as a negative control. A 50 mm diameter punch was used to cut an appropriate amount of bacterial cake from the edge of the strain and inoculated into the center of the drug-containing culture medium. The culture was inverted and incubated at 28°C in the dark. A culture medium without the added drug was also set up as a blank control. When the mycelial mat diameter of the blank control reached 60-70 mm, the mycelial diameter of each culture dish was measured using the cross-cross method. Three replicates were performed for each compound, and the results were averaged. The mycelial growth inhibition rate was calculated using the following formula to quantitatively evaluate the compound's antibacterial effect.

[0048] I(%)=[(Cd)-(Td)] / (Cd)×100

[0049] Where d is the well diameter (50 mm), C is the average colony diameter of the control group, and T is the average colony diameter of the treatment group. The EC values of the compound and the lead compound were determined and calculated using the Scatchard method. 50 value.

[0050] Table 1 The therapeutic effect of compound S1 on living potted plants of Rhizoctonia solani

[0051]

[0052] The results are shown in Table 1. The in vitro inhibitory activity of compound S1 against Rhizoctonia solani, tobacco leaf spot pathogen, and rice blast fungus was 100%. The EC values of compound S1 against Rhizoctonia solani, tobacco leaf spot pathogen, and rice blast fungus were 100%. 50 The values were 1.51mM, 1.53mM, and 1.55mM, respectively, indicating that compound S1 exhibited significant broad-spectrum antibacterial activity with good antibacterial activity. Further investigations were conducted using Rhizoctonia solani as a representative fungus to investigate the fungal control, therapeutic effects, and bactericidal mechanism of compound S1.

[0053] Example 3 Protective and therapeutic efficacy of compound S1 against detached leaves of rice sheath blight

[0054] The present invention uses a detached leaf method to systematically evaluate the control and treatment effects of the compound S1 on Rhizoctonia solani. The experiment selected rice plants cultured in a greenhouse for 60 days, collected fresh leaves and cut into uniform fragments of 12 cm. The compound S1 was dissolved in distilled water and formulated to 50mM, 100mM, and 200mM, with distilled water as a negative control and azoxystrobin (SC) as a positive control. To explore its control activity, the prepared agent solution was evenly sprayed on the surface of the rice leaves, and then the leaves were placed in a culture medium covered with filter paper, with three biological replicates set for each concentration. After the treated leaves were cultured in a light incubator for 24 hours, a Rhizoctonia solani cake with a diameter of 0.5 cm was inoculated in the center of the leaves and continued to be cultured for 2 days. The lesion area of each leaf was quantitatively analyzed using Image J software, and the control effect was calculated by the following formula to evaluate the inhibitory ability of the compound S1 on rice sheath blight.

[0055] Prevention and control effect (%) = (R0-R1) / (R1-0.81)×100

[0056] Among them, R0 and R1 are the average lesion areas of the blank control group and the treatment group, respectively. The area of the Rhizoctonia solani cake is 0.20 cm 2 .

[0057] To investigate therapeutic activity, first inoculate the center of a rice leaf with a cake of Rhizoctonia solani. 24 hours after infection, evenly spray the rice leaf with the prepared solution. The remaining steps are the same as above.

[0058] Results see Figures 1 to 3 As can be seen from the figure, the compound S1 has both preventive and therapeutic effects on rice sheath blight. Compared with the control group, as the concentration of the compound S1 increases, the leaf lesion area is significantly reduced. At concentrations of 50mM, 100mM and 200mM, the control effect of the compound S1 on detached leaves ( Figure 3 Figure A) and treatment effects ( Figure 3 Figure B) were 57.67% and 70.33%, 68.40% and 86.67%, 91.00% and 89.33% respectively. In comparison, the control effect of the positive control azoxystrobin at the same concentration ( Figure 3 Figure A) and treatment effects ( Figure 3 (Figure B) showed that the activity of compound S1 was significantly superior to that of the positive control, demonstrating its potential for controlling rice sheath blight. This result provides important evidence for further research into the in vivo mechanism of action of compound S1 and its practical application.

[0059] Example 4: Protective and therapeutic efficacy of compound S1 against rice sheath blight in living potted plants

[0060] The present invention selects rice variety Huayou 86 and cultivates it under greenhouse conditions for 60d. Subsequently, the cake of Rhizoctonia solani was inoculated in a 250mL conical flask containing 200mL potato dextrose broth (PDB) culture medium, and cultured in a dark environment for 5d with shaking to obtain mycelium and cut into small pieces of mycelial balls. In order to evaluate the control effect of the compound S1 on rice sheath blight, the experiment was carried out in the rice tillering stage. The compound S1 was dissolved and diluted to 50mM, 100mM, and 200mM with distilled water and evenly sprayed on the surface of the rice plants. Distilled water was used as a negative control, and azoxystrobin was used as a positive control. Three replicates were set for each group of experiments. After 24h of treatment, the mycelial balls were inoculated under the rice leaf sheath and immediately covered with aluminum foil to maintain humidity. The aluminum foil was removed after typical lesions appeared. After 7d of inoculation, the protective efficacy was calculated by the following formula to quantitatively evaluate the control effect of the compound S1. The formula is as follows:

[0061] Inhibition rate (%) = (P0-P 1) / P0×100

[0062] P0 and P1 represent the average diameters of lesions in the blank control group and the treated group, respectively.

[0063] To evaluate the therapeutic efficacy of compound S1 on rice plants, cultured Rhizoctonia solani pellets were first inoculated into the leaf sheaths of tillering rice plants and then wrapped in aluminum foil. After 24 hours, the entire plant was evenly sprayed with a solution of the desired concentration. The remaining steps and efficacy calculation formula were the same as above.

[0064] Table 2 Control effect of compound S1 on living potted plants of Rhizoctonia solani

[0065]

[0066] Table 3 The therapeutic effect of compound S1 on living potted plants of Rhizoctonia solani

[0067]

[0068] Results see Figure 4 and Figure 5 As shown in Tables 2 and 3, the compound S1 is significantly better than the positive control in both prevention and treatment effects. At a concentration of 200 mM, the prevention and treatment effects of the compound S1 were 87.27% and 85.12%, respectively, while the prevention and treatment effects of the positive control myclobutanil were 58.62% and 66.18%, respectively. In addition, the rice plants treated with the compound S1 did not show any toxic symptoms, indicating that it has high safety for rice. In summary, the compound S1 shows significant application potential as a fungicide, has a good prevention and control effect on rice sheath blight, and provides a new reference approach for its further development as an efficient and safe agricultural fungicide.

[0069] Example 5 Effect of Compound S1 on Sclerotium Formation of Rhizoctonia solani

[0070] First, the compound S1 was dissolved in distilled water, then added to the sterilized potato dextrose agar medium (PDA), prepared to 0.5mM, 1mM, and 2mM, and evenly poured into a 90mm culture dish, with three biological replicates for each concentration. The culture medium treated with equal volume of distilled water was used as a negative control, myclobutanil as a positive control, and an untreated PDA culture medium was set as a blank control. A 5mm diameter bacterial cake was inoculated in the center of the drug-containing PDA culture medium, cultured at 28°C in the dark for 14d, and then the sclerotia were collected. The collected sclerotia were dried at 60°C for 24h, weighed, and the sclerotia weight was recorded. The formula for calculating the sclerotia weight is as follows to quantitatively analyze the effect of the compound S1 on sclerotia formation. The formula is as follows:

[0071] Sclerotium formation inhibition rate (%) = [(W b -W t ) / W b ]×100

[0072] Among them, Wb and Wt are the average weights of sclerotia in the blank control and treatment groups, respectively.

[0073] Table 4 Inhibition rate of compound S1 on the formation of Rhizoctonia solani

[0074]

[0075] Results see Figure 6 As shown in Table 4, compound S1 significantly inhibits sclerotium formation, outperforming azoxystrobin, achieving an inhibition rate of 98.53% at a concentration of 2.0 mM. This reduces the occurrence of the disease at the source and prevents sclerotium formation in Rhizoctonia solani. Compared with the widespread use of traditional chemical pesticides, the precise use of compounds such as compound S1 that target sclerotium formation is expected to achieve a greener and more efficient agricultural disease control strategy.

[0076] Example 6 Effect of Compound S1 on Sclerotium Germination of Rhizoctonia solani

[0077] The present invention evaluates the effect of the compound S1 on sclerotium germination according to a standardized method. First, mature sclerotia were obtained from a culture medium cultured under dark conditions for 14 days. The compound S1 was formulated into 2mM, 4mM, and 8mM, and thoroughly mixed with sterilized potato dextrose agar (PDA) medium and poured evenly into a culture dish. Subsequently, 6 sclerotia were inoculated in each culture dish and incubated at a constant temperature of 28°C in the dark for 36 hours. The sclerotium germination inhibition rate was calculated by the following formula to quantitatively analyze the inhibitory effect of the compound S1 on sclerotium germination.

[0078] Sclerotium germination inhibition rate (%) = [(N b -N t ) / N b ]×100

[0079] Where N b and N t The numbers of sclerotia germination in the blank control and treatment groups are shown in Table 5. Three replicates were performed for each treatment.

[0080]

[0081] Results see Figure 7 As shown in Table 5, at the tested concentrations, azoxystrobin could not inhibit the germination of sclerotia, while compound S1 could effectively inhibit the germination of sclerotia at 8 mM.

[0082] Example 7 Observation of mycelial morphology after treatment with compound S1

[0083] The present invention uses scanning electron microscopy (SEM) technology to observe and analyze the samples based on the standardized method. First, the compound S1 is mixed with potato dextrose agar medium (PDA), prepared to the target concentration, and evenly poured into a 90mm culture dish. The PDA culture medium without adding the compound is used as a blank control (CK), and myclobutanil is used as a positive control. Subsequently, a 5mm diameter Rhizoctonia solani cake is inoculated in the center of the culture dish and cultured at a constant temperature of 28°C in the dark for 72 hours. After the culture is completed, a 5×5mm 2 Three bacterial blocks were randomly selected from each culture dish as samples. The bacterial blocks were placed in EP tubes containing 1 mL of 4% glutaraldehyde phosphate buffer, vacuumed with a syringe to remove the air in the tube, and fixed in a 4°C refrigerator overnight. After fixation, the samples were washed three times with 0.1 M phosphate buffer to completely remove residual glutaraldehyde. Subsequently, 0.5 mL of 1% osmium acid solution was added for secondary fixation for 90 minutes. The fixed samples were dehydrated in gradient ethanol (30%, 50%, 70%, 80%, 90%, 100%), each soaking for 10 minutes. After dehydration, the samples were dried using a critical point dryer and metal-coated. Finally, the treated samples were placed under a scanning electron microscope for observation to analyze their microscopic morphological characteristics.

[0084] Results see Figure 8 As can be seen in the figure, the mycelial surface structure of the blank control group was intact, with regular morphology and a smooth surface. However, the mycelial surface treated with compound S1 exhibited significant morphological abnormalities, manifesting as deformities, surface damage, and even breakage. This phenomenon suggests that compound S1 effectively inhibits fungal growth by disrupting the morphological and structural integrity of the fungal cell wall, leading to the leakage of cellular contents. This finding provides important morphological evidence for the bactericidal mechanism of compound S1 and lays the theoretical foundation for its further development as a cell wall-targeting antifungal agent.

[0085] Example 8 Determination of UV Absorption of Mycelium Treated with Compound S1

[0086] The present invention pre-treats the mycelial pellets of Rhizoctonia solani with reference to the method described in Experimental Example 3. First, 0.50 g of mycelium was accurately weighed and suspended in 0.01 M phosphate buffer (PBS). Subsequently, the compound S1 was added to PBS at a final concentration of 2 mM, 4 mM, and 8 mM, respectively. After thorough mixing, the mycelial suspension was placed on a shaker and cultured in the dark for 8 h. After the culture was completed, an appropriate amount of PBS suspension was transferred to a cuvette, and the absorbance was measured at 260 nm and 280 nm using an ultraviolet spectrophotometer to analyze the effect of the compound S1 on mycelial metabolism.

[0087] Results see Figure 9 As shown in the figure, compared with the blank control group, the absorbance values at 260nm and 280nm of the samples treated with compound S1 increased significantly with the concentration gradient, indicating a large amount of leakage of nucleic acids and proteins from the cells. This result directly proves that compound S1 exerts its bactericidal effect by destroying the structural integrity of the cell membrane, causing the outflow of cellular contents.

[0088] Example 9 Determination of the inhibition of mycelial enzyme activity by compound S1 treatment

[0089] This study aimed to evaluate the effects of compound S1 on the levels of three key enzymes involved in the biological respiratory chain: catalase (CAT), superoxide dismutase (SOD), succinate dehydrogenase (SDH), and lipid peroxide (LPO). Using Rhizoctonia solani as a model organism, the fungus was first activated in PDA medium for 2 days. A 5 mm diameter cake was then transferred to PDB medium and cultured on a shaker for 48 hours. Mycelia were treated with 0.25 mM, 2 mM, and 4 mM of compound S1, and the activities of CAT, SOD, and SDH, as well as LPO content, were measured according to the assay kit method.

[0090] Catalase (CAT) is a key enzyme widely found in plants, animals, microorganisms, and cultured cells. Its primary function is to scavenge H₂O₂, playing a crucial role in the scavenging system of reactive oxygen species (ROS). The specific assay steps are as follows: Mycelial tissue is collected and homogenized in an ice bath at a mass (g) to volume (V) ratio of 1:10. Centrifuge at 8000g at 4°C for 10 minutes, and the supernatant is placed on ice for analysis. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 405 nm, and zero with distilled water. Before measurement, incubate a 20 μmol / mL standard and reagent 1 in a 25°C water bath for 10 minutes. Add the sample according to the instructions, mix thoroughly, and let stand at room temperature for 10 minutes. Measure the absorbance at 405 nm in a 1 mL glass cuvette and record the absorbance as A₀, A₀ control, A₀ blank, and A₀ standard, respectively. Calculate ΔA₀ as: A₀ - A₀ control, ΔA₀ standard as: A₀ standard - A₀ blank, and ΔA₀ as: ΔA₀ standard - ΔA₀. The CAT activity was calculated as follows: CAT (U / g mass) = (ΔA / ΔA standard) × 20 × V standard / (V sample / V sample total × W) / T × F = 10 × (ΔA / ΔA standard) / W × F.

[0091] Superoxide dismutase (SOD) is a metalloenzyme widely distributed throughout the body. It catalyzes the dismutation of superoxide anions to produce H₂O₂ and O₂, playing a crucial role in antioxidant systems. The assay procedure is as follows: Mycelial tissue is collected and homogenized on ice at a mass (g) to extract volume (V) ratio of 1:10. Centrifuge at 8000g at 4°C for 10 minutes, and the supernatant is placed on ice for analysis. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 560 nm, and zero with distilled water. Immediately before use, incubate the working solutions of Reagents 1, 3, and 4 at 37°C for 5 minutes. Follow the sample addition instructions in the instruction manual, mix thoroughly, and incubate at 37°C for 30 minutes. Measure the absorbance at 560 nm in a 1 mL glass cuvette and record the absorbance as A₀, A₀, A₁₀, and A₂₀, respectively. Calculate ΔA₀ = A₀ - A₀, and ΔA₀ = A₁₀ - A₂₀. The formula for calculating SOD activity is: SOD (U / g mass) = [inhibition percentage / (1-inhibition percentage) × V total] / 1 mL / (W × V sample / V total sample) × F = 11.11 × inhibition percentage / (1-inhibition percentage) / W × F.

[0092] Succinate dehydrogenase (SDH) is a mitochondrial marker enzyme widely found in plants, animals, microorganisms, and cultured cells. Located in the inner mitochondrial membrane, it is a key enzyme connecting respiratory electron transport and oxidative phosphorylation. The assay steps are as follows: Weigh approximately 0.1 g of mycelial tissue, add 1 mL of Reagent 1 and 10 μL of Reagent 2, homogenize in an ice bath, centrifuge at 11,000 g and 4°C for 10 minutes, and place the supernatant on ice for testing. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 600 nm, and zero with distilled water. Preheat Reagent 3 at 25°C for 10 minutes before use. Follow the sample addition table in the instruction manual and immediately measure the initial absorbance A1 at a wavelength of 600 nm for 20 seconds. Then, react at 25°C for 5 minutes and measure the absorbance A2 at 5 minutes and 20 seconds. Calculate ΔA = A1 - A2 and record this as ΔA measurement and ΔA blank. The formula for calculating SDH activity is: SDH activity (U / g mass) = [(ΔA assay - ΔA blank) / (ε×d)×V anti total×109] / (V sample / V sample total×W) / T = 192.381×(ΔA assay - ΔA blank) / W.

[0093] Lipid peroxides (LPO) are peroxides formed by the action of unsaturated fatty acid chains on free radicals or reactive oxygen species. Elevated levels can damage cell structure and function and are closely associated with immunity and aging. The assay steps are as follows: Collect mycelial tissue and homogenize it on ice at a ratio of 1:10 (weight (g) to volume (V) of extract). Centrifuge at 8000g at 4°C for 10 minutes. Place the supernatant on ice for analysis. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelengths to 532nm and 600nm, and zero with distilled water. Prepare a 1000nmol / mL MDA standard solution and dilute it to 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625nmol / mL for later use. Follow the sample loading instructions. Incubate the mixture at 100°C in a water bath for 60 minutes, then cool it on ice. Centrifuge at 8000g at room temperature for 10 minutes. Pipette 900 μL of the supernatant into a 1 mL glass cuvette and measure the absorbance at 532 nm and 600 nm. Calculate ΔA = (A532 assay - A532 control) - (A600 assay - A600 control), and ΔA standard = (A532 standard - A532 blank) - (A600 standard - A600 blank). The formula for calculating LPO content is: LPO content (nmol / g mass) = X × V sample / (W × V sample / V sample total) = X / W.

[0094] Results see Figure 10 As can be seen from the figure, after treatment with the compound S1, the activities of CAT and SOD in Rhizoctonia solani increased significantly along the concentration gradient ( Figure 10 Figure A, Figure 10 Figure C), indicating that compound S1 may trigger the fungal defense response by inducing oxidative stress. However, compound S1 had no significant effect on the SDH activity in Rhizoctonia solani ( Figure 10 Figure B), indicating that SDH is not the target of compound S1. After treatment with compound S1, the LPO content in Rhizoctonia solani increased significantly ( Figure 10 D), and the degree of increase was positively correlated with the concentration of compound S1, indicating that compound S1 caused cell damage and functional destruction by inducing lipid peroxidation.

[0095] In summary, the compound S1 has a significant effect on the physiological functions of Rhizoctonia solani by regulating the activity of antioxidant enzymes and inducing lipid peroxidation, providing an important basis for elucidating its bactericidal mechanism.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of an amide compound or an agrochemically acceptable salt thereof in the preparation of an antifungal drug, characterized in that: The amide compound has the following structure:

2. The application according to claim 1, characterized in that The agrochemically acceptable salt includes hydrochloride.

3. The use according to claim 1 or 2, characterized in that: The fungi include one or more of Rhizoctonia solani, Pyricularia oryzae, and Magnaporthe grisea.

4. The use according to claim 1 or 2, characterized in that: The concentration of the amide compound or an agrochemically acceptable salt thereof is 0.1 to 300 mM.

5. The use according to claim 1 or 2, characterized in that: The amide compound or the agrochemically acceptable salt thereof has an antifungal effect by destroying the cell wall of the fungus.

6. The use according to claim 1 or 2, characterized in that: The amide compound or the agrochemically acceptable salt thereof plays an antifungal role by regulating the activity of antioxidant enzymes.

7. The use according to claim 1 or 2, characterized in that: The antioxidant enzyme is catalase and / or superoxide dismutase.

8. The use according to claim 1 or 2, characterized in that: The amide compound or the agrochemically acceptable salt thereof plays an antifungal role by inducing lipid peroxidation.

9. The use according to claim 1, characterized in that: The antifungal drug comprises an amide compound or an agrochemically acceptable salt thereof, and further comprises an agrochemically acceptable carrier and / or excipient.

10. The application according to claim 1, characterized in that: The dosage form of the antifungal drug is solution, emulsion, suspension, powder, foam, paste, granule or aerosol.

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