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. 2024 Apr 5;9(15):17297–17306. doi: 10.1021/acsomega.3c10294

Novel Flavonol Derivatives Containing 1,3,4-Thiadiazole as Potential Antifungal Agents: Design, Synthesis, and Biological Evaluation

Yuanxiang Zhou 1, Chenyu Gong 1, Zhiling Sun 1, Wei Zeng 1, Kaini Meng 1, Youshan An 1, Yuzhi Hu 1, Wei Xue 1,*
PMCID: PMC11024969  PMID: 38645355

Abstract

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In order to discover novel compounds with excellent agricultural activities, novel flavonol derivatives containing 1,3,4-thiadiazole were synthesized and evaluated for their antifungal activities. The bioassay results showed that some of the target compounds had good antifungal activities against Botrytis cinerea, Phomopsis sp. and Sclerotinia sclerotiorum in vitro. It is worth noting that the half-effective concentration (EC50) value of Y18 against B. cinerea was 2.4 μg/mL, which was obviously superior to that of azoxystrobin (21.7 μg/mL). The curative activity of Y18 at 200 μg/mL (79.9%) was better than that of azoxystrobin (59.1%), and its protective activity (90.9%) was better than that of azoxystrobin (83.9%). Morphological studies by using scanning electron microscopy and fluorescence microscopy revealed that Y18 could affect the normal growth of B. cinerea mycelium. In addition, the mechanism of action studies indicated that Y18 could affect the integrity of cell membranes by inducing the production of endogenous reactive oxygen species and the release of the malondialdehyde content, leading to membrane lipid peroxidation and the release of cell contents. The inhibitory activity of flavonol derivatives containing 1,3,4-thiadiazole on plant fungi is notable, offering significant potential for the development of new antifungal agents.

1. Introduction

Fungal plant pathogen infection causes significant losses to the global agricultural economy and poses a huge threat to human health and food security.1 Another notable example of a plant disease is gray mold, caused by the pathogen Botrytis cinerea. This organism has a broad host range and is notorious as one of the most devastating plant fungal diseases.2 This pathogen severely impairs the economic outcomes of various vital cash crops, such as grapes, strawberries, blueberries, and tomatoes. It can infect the roots, leaves, flowers, and fruits of plants, thereby reducing crop yields.3,4 At present, the use of fungicides in crop cultivation is among the most reliable strategies for controlling plant diseases.5 But the long-term and widespread use of chemical fungicides has led to an increase in the resistance of plant pathogenic fungi and posed severe threats to the environment.6,7 Therefore, the discovery of novel, efficient, and ecofriendly fungicides is indispensable to tackling the aforementioned challenge.

Flavonols, 3-hydroxyl flavones, are a unique flavonoid mainly found in dicotyledonous plants, especially in the flowers and leaves of some woody plants, such as the ginkgo biloba, sea buckthorn, and sophora flowers.8,9 Flavonoids, as important products of plant secondary metabolism, have various excellent physiological activities, extensive sources, minimal side effects, and exceptional safety.10 Moreover, flavonols and their derivatives have good antiviral,11,12 antibacterial,13,14 insecticidal,15,16 antioxidant,17 anticancer,18,19 and other biological activities,20 and are widely used in the fields of pesticides and pharmaceuticals.

In addition, 1,3,4-thiadiazole and its derivatives, a category of five-membered heterocyclic compounds, possess good physical and chemical properties.21 Their structural units contain the basic structural framework of carbon, nitrogen and sulfur, which may be chemically modified to obtain highly efficient and low-toxicity bioactive derivatives.22,23 Among them, 1,3,4-thiadiazole derivatives have a wide variety of biological activities, such as antiviral,24 antibacterial,25,26 antifungal,27,28 insecticidal,29,30 and other biological activities.31,32 Their vast applications in pesticides have been acknowledged widely. Currently, among the various successfully developed products are the bactericides bismerthiazol, thiodiazole copper, and the herbicide fluthiamide (Figure 1).

Figure 1.

Figure 1

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Chemical structures of 1,3,4-thiadiazole fragments with biological activities.

In summary, we propose to introduce substituting 1,3,4-thiadiazole groups into the structure of flavonols by active substructure splicing to synthesize a series of flavonol derivatives containing 1,3,4-thiadiazole. Following biological activity screening and mechanism studies, the compound Y18 was discovered to possess superior antifungal activity compared to azoxystrobin, which provides significant potential for the development of novel antifungal drugs (Figure 2).

Figure 2.

Figure 2

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Design of target compounds.

2. Results and Discussion

2.1. Synthesis

Scheme 1 outlines the design and synthesis of 22 flavonol derivatives containing 1,3,4-thiadiazole (Y1–Y22). First, by using substituted thionylhydrazine and CS2 as raw materials, intermediates 1 were obtained in the reflux reaction. Utilizing substituted 2-hydroxyacetophenone and substituted benzaldehyde as raw materials, EtOH was used as a solvent to obtain intermediates 2 by a hydroxyl aldehyde condensation reaction under alkaline conditions at room temperature. Then, using intermediates 2 as the raw material, MeOH as the solvent, and H2O2 as the oxidant, intermediate 3 was obtained by cyclization reaction under alkaline conditions. 1,3-dibromopropane reacted with intermediates 3 to synthesize intermediates 4 at room temperature. Finally, intermediates 4 and 1 were stirred at 25 °C for 8 h with K2CO3 as a weak acid binder to yield flavonol derivatives containing 1,3,4-thiadiazole. The structures of these compounds were verified by 1H, 13C, and 19F nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) data. Refer to the Supporting Information for detailed synthesis steps.

Scheme 1. Synthetic Route of the Target Compounds Y1–Y22.

Scheme 1

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2.2. Evaluation of Antifungal Activity

2.2.1. Antifungal Activity In Vitro

Preliminary antifungal activity results of Y1–Y22 against ten types of pathogenic fungi in vitro are presented in Table 1. The data illustrated that some compounds exhibit excellent antifungal activities. The inhibition rates of Y12, Y13, Y17, and Y18 against B. cinerea were 91.7, 83.2, 87.3, and 96.2%, respectively, which were superior to that of azoxystrobin (80.7%). The inhibition rates of Y14 and Y21 against Phomopsis sp. were 73.8 and 72.8%, respectively, which were superior to those of azoxystrobin (58.1%). The antifungal efficacy of Y14 against Sclerotinia sclerotiorum was 71.5%, which was similar to that of azoxystrobin (71.9%).

Table 1. Inhibition Effect of Target Compounds against Ten Plant Phytopathogenic Fungi at 100 μg/mL.
compounds inhibition rates (%)a
  Bcb Ps Cg Rs Pc Ss Fcu Fg Ab Fca
Y1 55.5 ± 1.2 32.2 ± 3.0 47.7 ± 1.7 54.0 ± 3.9 30.0 ± 3.7 40.6 ± 1.1 20.5 ± 1.1 33.2 ± 3.0 44.4 ± 1.6 44.4 ± 0.6
Y2 61.3 ± 3.8 40.4 ± 4.1 46.0 ± 1.2 55.5 ± 3.0 38.4 ± 1.2 38.2 ± 1.1 18.9 ± 1.1 36.5 ± 2.2 46.0 ± 2.9 33.9 ± 1.1
Y3 65.1 ± 3.3 37.2 ± 1.1 47.7 ± 1.7 59.3 ± 4.0 34.5 ± 3.2 63.6 ± 1.7 32.3 ± 1.7 24.9 ± 0.9 51.7 ± 1.3 48.8 ± 0.9
Y4 36.6 ± 0.9 34.1 ± 1.1 42.7 ± 1.4 38.6 ± 3.0 41.8 ± 3.8 43.8 ± 3.4 35.0 ± 1.2 38.5 ± 2.4 51.0 ± 4.1 40.3 ± 1.1
Y5 38.7 ± 2.8 60.2 ± 1.1 41.8 ± 2.2 43.8 ± 4.9 36.3 ± 2.2 44.6 ± 2.7 32.7 ± 1.2 31.6 ± 3.6 49.8 ± 1.6 42.3 ± 0.9
Y6 60.1 ± 3.9 51.7 ± 1.3 32.2 ± 2.0 48.5 ± 2.4 28.7 ± 4.4 10.7 ± 1.4 41.7 ± 1.1 32.8 ± 3.3 35.6 ± 1.3 42.3 ± 2.6
Y7 74.6 ± 2.7 36.8 ± 1.1 30.1 ± 2.6 55.3 ± 1.0 32.1 ± 2.7 51.2 ± 3.7 29.1 ± 1.4 35.7 ± 1.7 32.6 ± 1.1 39.1 ± 1.7
Y8 62.2 ± 3.5 53.5 ± 2.8 35.6 ± 1.9 51.5 ± 0.8 31.2 ± 4.9 52.6 ± 2.3 30.7 ± 1.1 28.3 ± 2.6 42.9 ± 0.9 42.7 ± 1.8
Y9 54.4 ± 0.9 55.0 ± 4.6 36.0 ± 1.2 52.2 ± 1.0 49.4 ± 2.9 53.8 ± 1.6 31.1 ± 1.6 31.6 ± 3.8 39.8 ± 0.9 47.6 ± 1.1
Y10 56.7 ± 2.2 37.2 ± 1.1 38.9 ± 1.2 47.1 ± 1.8 51.9 ± 2.9 45.0 ± 1.7 30.3 ± 2.2 33.2 ± 4.3 28.7 ± 1.3 41.5 ± 2.6
Y11 33.3 ± 4.7 27.1 ± 1.7 24.3 ± 1.7 57.4 ± 1.8 35.7 ± 1.2 45.7 ± 1.7 46.5 ± 2.6 33.5 ± 1.7 36.8 ± 1.7 47.6 ± 1.1
Y12 91.7 ± 1.2 59.6 ± 1.9 36.0 ± 1.2 55.5 ± 2.0 24.5 ± 2.7 54.2 ± 1.4 37.8 ± 1.1 35.7 ± 1.7 39.5 ± 1.1 45.6 ± 3.0
Y13 83.2 ± 2.4 42.8 ± 1.1 41.8 ± 1.7 54.0 ± 2.0 34.2 ± 3.8 47.0 ± 1.4 46.5 ± 1.1 35.7 ± 1.7 39.8 ± 1.6 52.4 ± 1.1
Y14 69.1 ± 1.9 73.8 ± 1.7 54.0 ± 1.2 65.6 ± 1.6 16.8 ± 3.4 71.5 ± 1.2 40.9 ± 1.4 20.2 ± 1.6 47.9 ± 1.1 50.8 ± 1.8
Y15 58.8 ± 2.4 59.0 ± 0.9 36.0 ± 1.2 40.1 ± 0.8 32.9 ± 3.8 57.4 ± 0.9 39.4 ± 1.1 51.6 ± 3.8 34.5 ± 1.1 47.6 ± 1.1
Y16 50.4 ± 4.2 50.2 ± 2.2 23.9 ± 1.2 40.8 ± 2.4 32.5 ± 4.5 48.8 ± 1.8 39.4 ± 1.1 31.2 ± 2.0 30.3 ± 1.1 51.6 ± 2.0
Y17 87.3 ± 1.1 66.1 ± 0.5 46.4 ± 1.2 65.2 ± 2.0 22.4 ± 4.4 65.3 ± 2.8 34.3 ± 1.6 28.4 ± 1.7 48.3 ± 1.1 50.4 ± 1.2
Y18 96.2 ± 1.7 65.3 ± 1.1 45.2 ± 1.7 63.4 ± 2.4 22.4 ± 2.9 58.7 ± 2.3 29.5 ± 0.9 18.0 ± 1.6 47.1 ± 1.3 51.2 ± 0.9
Y19 60.1 ± 4.3 49.4 ± 2.9 41.0 ± 5.7 45.6 ± 1.0 28.3 ± 4.0 47.0 ± 1.4 41.3 ± 0.9 32.0 ± 2.3 40.2 ± 1.9 46.4 ± 1.7
Y20 65.9 ± 4.1 42.8 ± 3.5 34.3 ± 1.7 45.2 ± 2.4 49.8 ± 3.4 45.0 ± 09 26.0 ± 1.1 26.6 ± 2.2 31.4 ± 1.6 46.4 ± 0.9
Y21 58.4 ± 2.6 72.8 ± 0.9 45.2 ± 4.6 46.3 ± 1.6 46.8 ± 2.0 66.9 ± 3.4 35.4 ± 1.1 44.3 ± 3.3 42.5 ± 1.3 48.4 ± 1.1
Y22 52.7 ± 4.3 38.0 ± 4.3 37.7 ± 0.9 52.8 ± 1.0 52.7 ± 4.3 41.7 ± 3.2 28.7 ± 0.9 37.7 ± 0.9 36.8 ± 1.7 44.4 ± 1.2
AZc 80.7 ± 1.2 58.1 ± 1.1 56.1 ± 2.4 75.7 ± 0.3 75.5 ± 1.9 71.9 ± 2.3 48.0 ± 1.9 36.6 ± 3.4 24.5 ± 0.9 60.9 ± 0.9
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a

Average of three replicates.

b

Rs (Rhizoctonia solani), Bc (B. cinerea), Fg (Fusarium graminearum), Cg (Colletotrichum gloeosporioides), Ss (S. sclerotiorum), Pc (Phytophthora capsica), Ab (Alternaria brassicae), Fcu (Fusarium oxysporum f. sp. cucumerinum), Fca (Fusarium oxysporum f. sp. capsicum), and Ps (Phomopsis sp.).

c

AZ (azoxystrobin).

By evaluating the EC50 values of compounds with good activity against three fungal strains, the antifungal potential of these synthetic compounds was further assessed (Table 2 and Figure 3). It is noteworthy that the EC50 value of Y18 against B. cinerea was 2.4 μg/mL, which was obviously superior to that of azoxystrobin (21.7 μg/mL), and the EC50 value of Y14 against Phomopsis sp. was 21.2 μg/mL and better than that of azoxystrobin (29.2 μg/mL).

Table 2. EC50 Values of Several Target Compoundsa.
fungi compounds EC50 (μg/mL) r2 regression equation
Bc Y12 3.5 0.9826 y = 1.2119x + 4.3468
  Y13 4.8 0.9966 y = 0.7208x + 4.5068
  Y17 7.2 0.9691 y = 1.0206x + 4.1271
  Y18 2.4 0.9841 y = 1.0797x + 4.5941
  AZ 21.7 0.9727 y = 1.0730x + 3.5651
Ss Y14 45.9 0.9661 y = 1.1608x + 3.0713
  AZ 43.6 0.9666 y = 0.8361x + 3.6291
Ps Y14 21.2 0.9503 y = 0.9175x + 3.7831
  Y21 25.5 0.9514 y = 0.9474x + 3.6677
  AZ 29.2 0.9953 y = 0.5431x + 4.2039
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a

Average of three replicates.

Figure 3.

Figure 3

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Antifungal activities of Y18 against B. cinerea (Bc) and Y14 against Phomopsis sp. (Ps) in vitro.

2.2.2. Structure–Activity Relationship

First, the antifungal activity was higher when the R1 group was substituted with –H rather than –NH2. Second, compounds with electron-withdrawing groups on the phenyl substituent of the flavonol structure had better antifungal activity, specifically, Y18 (3-F) > Y12 (4-CH3) > Y13 (4-OCH3). Finally, the antifungal activity was greater when R3 was 3-F compared to when R3 was 4-F. In summary, the antifungal activity of Y18 (R1 = H, R2 = H, and R3 = 3-F) was significantly superior to other target compounds and azoxystrobin.

2.2.3. Antifungal Activity of Y18 and Y14 In Vivo

Y18 was chosen to further explore the antifungal activity against B. cinerea on blueberry leaves in vivo. As depicted in Table 3 and Figure 4, compared with the untreated negative control group, the leaf spot length of blueberries treated with Y18 was smaller and the leaf decay was lighter, indicating that Y18 obviously had inhibitory activity against B. cinerea. The curative activity of Y18 (79.9%) at 200 μg/mL was superior to that of azoxystrobin (59.1%), and the protective activity (90.9%) was better than that of azoxystrobin (83.9%). The experimental results suggested that Y18 could be potentially utilized in crop protection.

Table 3. Curative and Protective Activities of Y18 against B. cinerea In Vivoa.
treatment concentration (μg/mL) curative activity
 protective activity
    lesion length (mm) control efficacy (%) lesion length (mm) control efficacy (%)
Y18 200 10.0 ± 1.7c 79.9 7.2 ± 0.8c 90.9
AZ 200 15.2 ± 3.0b 59.1 8.8 ± 1.0b 83.9
negative control   29.8 ± 4.8a   28.8 ± 4.2a  
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a

Values are the mean ± SD of three replicates. Statistical analysis was conducted using SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

Figure 4.

Figure 4

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Curative and protective activity of Y18 and azoxystrobin against B. cinerea on blueberry leaves.

Y14 was selected to evaluate its antifungal activity against Phomopsis sp. on kiwifruit further in vivo. As can be seen in Table 4 and Figure 5, the protective activity of Y14 against Phomopsis sp. was 71.8% at 200 μg/mL, and its curative activity was 60.2%, which was superior to those of azoxystrobin (60.2 and 59.9%, respectively). It can be shown that Y14 has excellent antifungal activity against Phomopsis sp. in vivo.

Table 4. Curative and Protective Activities of Y14 against Phomopsis sp. In Vivoa.
treatment concentration (μg/mL) curative activity
 protective activity
    lesion length (mm) control efficacy (%) lesion length (mm) control efficacy (%)
Y14 100 34.8 ± 1.2c 37.0 25.8 ± 0.8c 57.5
  200 23.8 ± 0.8d 60.2 18.8 ± 1.7d 71.8
AZ 100 38.5 ± 1.1b 29.2 32.5 ± 1.1b 43.9
  200 24.0 ± 1.3d 59.9 24.5 ± 0.8c 60.2
negative control   52.3 ± 3.4a   54.0 ± 3.4a  
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a

Values are mean ± SD of three replicates. Statistical analysis was conducted using SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

Figure 5.

Figure 5

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Curative and protective activity of Y14 and azoxystrobin against Phomopsis sp. on kiwifruit.

2.3. Morphological Analysis by Scanning Electron Microscopy and Fluorescence Microscopy

The effect of Y18 on the B. cinerea mycelium was assessed by scanning electron microscopy (SEM), which revealed morphological changes. As depicted in Figure 6, the mycelium of the untreated negative control group had a full shape, smooth surface, and vigorous growth, whereas it was wrinkled, collapsed, and severely deformed after being treated with 50 and 100 μg/mL Y18, respectively. The study revealed that with increasing Y18 concentrations, the damage to the mycelium became more severe, resulting in adverse effects on its normal growth.

Figure 6.

Figure 6

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SEM images of the hyphae of B. cinerea after treatment with different concentrations of Y18. (A) 0 μg/mL, (B) 50 μg/mL, and (C) 100 μg/mL. Scale for 20 μm.

Propyl iodide (PI) is a unique nuclear dye that emits red fluorescence when embedded in double-stranded DNA, and is primarily used for DNA staining. When more and brighter red fluorescence appears, it indicates more severe cell membrane damage.33 As depicted in Figure 7, the blank control group displayed almost no fluorescence, indicating that the mycelium within this group was undamaged. However, with the increase of concentration of Y18, more red fluorescence was present, indicating that Y18 can damage the integrity of the mycelial cell membrane.

Figure 7.

Figure 7

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Morphological observation of B. cinerea after treatment with Y18 at different concentrations by fluorescence microscopy (FM). (A1–D1) Under bright field, (A2–D2) Under a fluorescence field, (A1,A2) 0 μg/mL, (B1,B2) 25 μg/mL, (C1,C2) 50 μg/mL, and (D1,D2) 100 μg/mL. Magnification 100 × 10. Scale for 10 μm.

2.4. Reactive Oxygen Species Assay of B. cinerea

Reactive oxygen species (ROS) is important to the regulation of a normal cellular process. ROS imbalance can cause the body’s oxidation-antioxidant imbalance, resulting in cell membrane destruction and cell death.34,35 As depicted in Figure 8, the untreated negative control group demonstrated almost no green fluorescence. However, after treatment with various concentrations of Y18, it was observed that the fluorescence intensity rose with increasing concentration, showing that Y18 can stimulate the production of ROS and cause an oxidation-antioxidant imbalance in the body.

Figure 8.

Figure 8

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Effects on the ROS of B. cinerea treated with Y18 at different concentrations. (A1–C1) Under a bright field, (A2–C2) under a fluorescence field, (A1,A2) 0 μg/mL, (B1,B2) 12.5 μg/mL, and (C1,C2) 25 μg/mL. Magnification 100 × 10. Scale for 10 μm.

2.5. Spore Germination Assay of B. cinerea

The inhibitory effect on B. cinerea spore growth can substantially reduce damage to the host plant. As depicted in Figure 9, the relative inhibition rates of Y18 on spore germination at concentrations of 200, 100, 50, 25, and 12.5 μg/mL were 98.2, 92.7, 73.7, 67.3, and 26.7%, respectively, and the EC50 value was 21.1 μg/mL. The results revealed that Y18 was capable of inhibiting the germination of spores effectively.

Figure 9.

Figure 9

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Effects of Y18 on spore germination of B. cinerea for different concentrations. (A) 0 μg/mL, (B) 12.5 μg/mL, (C) 25 μg/mL, (D) 50 and μg/mL, (E) 100 μg/mL, and (F) 200 μg/mL. Magnification 10 × 10. Scale for 10 μm. Statistical analysis was conducted using SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

2.6. Cell Membrane Permeability Assay of B. cinerea

Cell membranes are crucial for the maintenance of cell shape, structural integrity, and physiological function. The permeation of cell membranes can be assessed through the determination of relative conductivity.36 As depicted in Figure 10, when Y18 was applied to the mycelium, the relative conductivity of the mycelium exhibited a significant increase, positively correlating with the increasing drug concentration. The upward trend in relative conductivity was notably greater than that of the untreated negative control group. The experimental results indicated that Y18 was capable of destroying the cell membrane’s structure, enhancing its permeability, and culminating in the death of mycelia.

Figure 10.

Figure 10

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Changes in cell membrane permeability of Y18 against B. cinerea. Statistical analysis was conducted using SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

2.7. Cytoplasmic Leakage Assay of B. cinerea

The absorbance of mycelium suspensions at 260 and 280 nm was measured using an ultraviolet–visible spectrophotometer to investigate the leakage of cytoplasmic substances such as nucleic acids and proteins.37 From Figure 11, it is clear that the absorbance values after treatment with varying concentrations of Y18 increased significantly with increasing drug concentration as compared to that of the untreated negative control group. The conclusion drawn from this is that Y18 caused a notable release of nucleic acids and proteins from the mycelium.

Figure 11.

Figure 11

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Release of cellular contents from B. cinerea after treatment with Y18. Statistical analysis was conducted using SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

2.8. Malondialdehyde Content Assay of B. cinerea

The malondialdehyde (MDA) content is a crucial metabolite in the peroxidation process of biological cell membranes. An increase in MDA levels, an indicator of oxidative damage, may suggest the extent of membrane damage and thus the severity of the cell damage.38 When compared with the untreated negative control group, it can be seen that the level of the MDA content gradually increased as the Y18 concentration increased following treatment with varying concentrations, as depicted in Figure 12. Consequently, Y18 is capable of notably increasing the level of lipid peroxidation in the cell membrane of the B. cinerea mycelium, thereby inducing cell membrane damage, which is in line with the previous experimental analysis.

Figure 12.

Figure 12

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MDA contents of B. cinerea treated with Y18. Statistical analysis was conducted by SPSS 27.0 software. Different letters indicate significant differences at p < 0.05 in the same group.

3. Conclusions

In summary, 22 flavonol derivatives containing 1,3,4-thiadiazole were designed and synthesized, and the structures of all the target compounds were determined by NMR and HRMS. The results of antifungal experiments in vitro showed that Y18 had good antifungal activity against B. cinerea, and its EC50 value was 2.4 μg/mL, which was obviously superior to that of azoxystrobin (21.7 μg/mL). Moreover, the activity experiment results in vivo showed that the curative activity of Y18 (79.9%) at 200 μg/mL was better than that of azoxystrobin (59.1%), and the protective activity (90.9%) was better than that of azoxystrobin (83.9%). Y18 also can effectively inhibit conidial germination to reduce damage to the host plant. Preliminary studies on the mechanism indicated that Y18 could affect the integrity of cell membranes by inducing endogenous ROS production, causing lipid peroxidation of cell membranes, and releasing cell contents, and the specific mechanism of action of Y18 with B. cinerea is under further exploration.

4. Materials and Methods

4.1. Instruments and Chemicals

1H, 13C, and 19F NMR spectra were determined by a Bruker 400 NMR spectrometer (Bruker Corporation, Germany). HRMS data were obtained by a Thermo Scientific Q Exactive instrument (Thermo Scientific, America). SEM data were measured on an FEI Nova Nano 450 (Hillsboro, OR, America). An Olympus-BX53 fluorescence microscope and a CX21FS1 microscope were obtained from Olympus Ltd., Japan. The reagents and solvents utilized in this study are of analytical grade, and the kits employed are manufactured by Beijing Solaibao Technology Co., Ltd.

4.2. Fungi

R. solani, B. cinerea, F. graminearum, C. gloeosporioides, S. sclerotiorum, P. capsica, A. brassicae, Fusarium oxysporumf. sp. cucumerinum (F. sp.cucumerinum), Fusarium oxysporum f. sp. capsicum (F. sp. capsicum), and Phomopsis sp. were selected for the experiment. These fungi were cultivated on potato dextrose agar plates at 25 ± 1 °C and preserved at 4 °C.

4.3. Synthesis

4.3.1. General Procedure for the Synthesis of Intermediates 1–4

Intermediates 1–4 were synthesized according to the reported procedures.39−42 Refer to the Supporting Information for detailed synthesis steps.

4.3.2. General Procedure for the Synthesis of Target Compounds Y1–Y22

Intermediates 1 (1.61 mmol), K2CO3 (2.01 mmol), and 20 mL of DMF were added sequentially to a 50 mL round-bottomed flask and stirred at room temperature for 30 min. Then, intermediates 4 (1.34 mmol) were added slowly and reacted for 10–12 h at 25 °C. The reaction was monitored by TLC (petroleum ether/ethyl acetate = 2:1, v/v), and when the reaction was completed, the system was extracted with ethyl acetate. The organic layer was obtained by layering, and the solvent was removed to obtain the crude product. Finally, the target compounds Y1–Y22 were isolated by column chromatography, where the crude product was eluted with a (petroleum ether/ethyl acetate = 5:1, v/v) gradient.

4.4. Bioassays

4.4.1. Antifungal Activity In Vitro

The inhibitory activity of Y1–Y22 against ten phytopathogenic fungi was tested according to refs (43–46). Refer to the Supporting Information for detailed steps. Based on the preliminary test results, the compounds with promising antifungal activity were identified by the EC50 value.

4.4.2. Antifungal Activity In Vivo

The in vivo antifungal test was performed according to the results of the in vitro antifungal activity assay. The test method is determined according to the methods reported in the literature.47 Refer to the Supporting Information for detailed steps.

4.5. Antifungal Mechanism Study

4.5.1. Effects of Y18 Treatment on Cell Membrane Integrity

4.5.1.1. Morphological Analysis by SEM

First, the potato dextrose broth (PDB) medium containing the mycelia of B. cinerea was cultured at 28 °C and 180 rpm for 24 h. Then, Y18 solution (50 and 100 μg/mL) was added and incubated with mycelium at 28 °C and 180 rpm. Subsequently, mycelium was washed with 0.01 mol/L phosphate-buffered saline (PBS) and then maintained for 24 h by adding 1.5 mL of a 2.5% glutaraldehyde solution. Finally, the mycelium was washed with ethanol and freeze-dried to make samples.48

4.5.1.2. Morphological Analysis by FM

As abovementioned, the mycelium was cleaned three times with PBS, and then 10 μL of PI solution (10 mg/L) was added to stain the mycelium. The mycelium was incubated at 37 °C for 25 min and then washed with PBS three times. Some mycelium was taken on a slide, then a droplet of glycerin was added, and the cover slide was covered to make a sample and to be observed.49,50

4.5.2. Determination of ROS

The mycelium of B. cinerea was prepared by following the procedure outlined in Section 4.5.1. Y18 solution (12.5 and 25 μg/mL) was added and incubated with mycelium for 24 h, and the mycelium was washed with PBS three times. Subsequently, the hyphae were treated with 0.1 mL of DCFH-DA (25 μmol/L) and then incubated in a dark environment at 37 °C for 60 min. This was followed by three washes with PBS. Finally, samples were prepared and observed by FM.51

4.5.3. Determination of Spore Germination

The mycelium of B. cinerea was prepared following the procedure outlined in Section 4.5.1. The PDB medium containing B. cinerea was cultured at 28 °C for 7 days. After a large number of spores were formed, a spore suspension (1.5 × 105 spores/mL) was produced with 0.1% Tween 80 solution.52 Refer to the Supporting Information for detailed steps.

4.5.4. Determination of Cell Membrane Permeability

The mycelium of B. cinerea was prepared following the procedure outlined in Section 4.5.1. First, the mycelium was washed with sterile water and then filtered. Then, 200 mg of mycelium was weighed and treated with Y18 (12.5, 25, and 50 μg/mL). Then, the conductivity was measured for 0, 30, 60, 90, 120, 150, 180, and 240 min. Finally, after boiling the mycelium for 1 h, the conductivity was measured after cooling.53

4.5.5. Determination of Cytoplasmic Content Leakage

The mycelium of B. cinerea was prepared following the procedure outlined in Section 4.5.1. 100 mg of mycelium was weighed and treated with Y18 (6.25, 12.5, and 25 μg/mL), cultured at 25 °C for 10 h. Finally, the absorbance of the supernatant was measured at 260 and 280 nm on an ultraviolet–visible spectrophotometer.54

4.5.6. Determination of MDA Contents

The literature indicates that the MDA contents can reflect the extent of cell membrane damage by oxidative stress.55 The mycelium of B. cinerea was prepared following the procedure outlined in Section 4.5.1. Y18 (0, 12.5, 25, 50, and 100 μg/mL) was added to the cultured mycelium and incubated at 28 °C. After 24 h, the mycelium was washed with sterile water and then filtered. Finally, the mycelium was freeze-dried for 2 h, and the MDA contents were determined according to the kit instructions.

Acknowledgments

This research was completed with the support of the National Natural Science Foundation of China (no. 32072446) and the Science Foundation of Guizhou Province (no. 20192452).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c10294.

  • Characterization data, 1H, 13C, and 19F NMR spectra, and HRMS of title compounds Y1–Y22 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao3c10294_si_001.pdf (5.9MB, pdf)

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