Fungicidal Activity of New Pyrrolo[2,3-d]thiazoles and Their Potential Action on the Tryptophan Metabolic Pathway and Wax Biosynthesis.

The main challenge in the development of agrochemicals is the lack of new leads and/or targets. It is critical to discover new molecular targets and their corresponding ligands. YZK-C22, which contains a 1,2,3-thiadiazol-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole skeleton, is a fungicide lead compound with broad-spectrum fungicidal activity. Previous studies suggested that the [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole scaffold exhibited good antifungal activity. Inspired by this, a series of pyrrolo[2,3-d]thiazole derivatives were designed and synthesized through a bioisosteric strategy. Compounds C1, C9, and C20 were found to be more active against Rhizoctonia solani than the positive control YZK-C22. More than half of the target compounds provided favorable activity against Botrytis cinerea, where the EC50 values of compounds C4, C6, C8, C10, and C20 varied from 1.17 to 1.77 µg/mL. Surface plasmon resonance and molecular docking suggested that in vitro potent compounds C9 and C20 have a new mode of action instead of acting as pyruvate kinase inhibitors. Transcriptome analysis revealed that compound C20 can impact the tryptophan metabolic pathway, cutin, suberin, and wax biosynthesis of B. cinerea. Overall, pyrrolo[2,3-d]thiazole is discovered as a new fungicidal lead structure with a potential new mode of action for further exploration.

Design, synthesis and systemic acquired resistance of 2-benzothiadiazolylquinoline-4-carboxamides by COI1 based virtual screening.

Coronatine-insensitive 1 (COI1) has been identified as a target receptor of plant elicitor coronatine (COR). To discover novel plant elicitor leads, most of the potential molecules among 129 compounds discovered from the ZINC database by docking based virtual screening targeting COI1 were quinoline amides. On this lead basis, 2-benzothiadiazolylquinoline-4-carboxamides were rationally designed and synthesized for bioassay. All target compounds did not show significantly in vitro antifungal activity, compounds 4d, 4e and 4o displayed good in vivo systemic acquired resistance activity for Arabidopsis thaliana against Hyaloperonospora arabidopsidis isolate Noco2 with over 80% of inhibitory rate at the concentration of 50 µM. These results indicate that 2-benzothiadiazolylquinoline-4-carboxamides are promising plant elicitor leads for further study.

Discovery of Trisubstituted N-Phenylpyrazole Containing Diamides with Improved Insecticidal Activity.

To increase the structural diversity of insecticides and meet the needs of effective integrated insect management, the structure of chlorantraniliprole was modified based on a previously established three-dimensional quantitative structure-activity relationship (3D-QSAR) model. The pyridinyl moiety in the structure of chlorantraniliprole was replaced with a 4-fluorophenyl group. Further modifications of this 4-fluorophenyl group by introducing a halogen atom at position 2 and an electron-withdrawing group (e.g., iodine, cyano, and trifluoromethyl) at position 5 led to 34 compounds with good insecticidal efficacy against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compound IV f against M. separata showed potency comparable to that of chlorantraniliprole. IV p against P. xylostella displayed a 4.5 times higher potency than chlorantraniliprole. In addition, IV d and chlorantraniliprole exhibited comparable potencies against S. frugiperda. Transcriptome analysis showed that the molecular target of compound IV f is the ryanodine receptor. Molecular docking was further performed to verify the mode of action and insecticidal activity against resistant P. xylostella.

Computation-Directed Molecular Design, Synthesis, and Fungicidal Activity of Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are a class of fungicides targeting the pathogenic fungi mitochondrial SDH. Here, molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular dynamics (MD) simulations were used to guide SDHI innovation. Molecular docking was performed to explore the binding modes of SDH and its inhibitors. 3D-QSAR models were carried out on 33 compounds with activity against Rhizoctonia cerealis (R. cerealis); their structure-activity relationships were analyzed using comparative molecular field analysis and comparative molecular similarity indices analysis. MD simulations were used to assess the stability of the complexes under physiological conditions, and the results were consistent with molecular docking. Binding free energy was calculated through the molecular mechanics generalized born surface area method, and the binding free energy was decomposed. The results are consistent with the activity of bioassay and indicate that van der Waals and lipophilic interactions contribute the most in the molecular binding process. Afterward, we designed and synthesized 12 compounds under the guidance of the above-mentioned analyses, bioassay found that F9 was active against R. cerealis with the EC50 value of 9.43 µg/mL, and F4, F5, and F9 were active against Botrytis cinerea with an EC50 values of 5.80, 3.17, and 1.63 µg/mL, respectively. They all showed good activity between positive controls of pydiflumetofen and thifluzamide. Our study provides new considerations for effective SDHIs discovery.

Synthesis of fungicidal morpholines and isochromenopyridinones via acid-catalyzed intramolecular reactions of isoindolinones.

Acid-catalyzed intramolecular cyclization or rearrangement of isoindolinone derivatives is described. 3-Hydroxy/ethoxy-3,4-dihydro-6H-[1,4]-oxazino-[3,4-a]-isoindol-6-ones are obtained in moderate to good yields. Further acid-catalyzed intramolecular rearrangement reactions give 6H-isochromeno-[4,3-b]-pyridin-6-ones. The mild reaction conditions with convenient starting materials show broad substrate scope and provide the target compounds as novel pesticide leads with good fungicidal or systemical acquired resistance activities.

Design and Synthesis of Pyrimidine Amine Containing Isothiazole Coumarins for Fungal Control.

Developing new fungicides is always crucial to protecting crops. A series of 4-(3,4-dichloroisothiazol-5-yl)-7-(2-((5-(5-pyrimidin-4-yl)amino)ethoxy)-8-methyl) coumarin derivatives were designed and synthesized by Williamson ether condensation and substitution reactions. Structure determinations were clarified by 1H NMR, 13C NMR, and HRMS, and compound 4h crystallized by the fusion method for further structural confirmation. The in vitro bioassay results showed that the target compounds displayed good fungicidal activity against Alternaria solani, Botrytis cinerea, Cercospora arachidicola, Fusarium graminearum, Physalospora piricola, Rhizoctonia solani, and Sclerotinia sclerotiorum. Among them, compounds 4b and 4d showed higher inhibitory activity against R. solani, with EC50 values of 11.3 and 13.7 µg/mL, respectively, and they were more active than the positive control diflumetorim with an EC50 value of 19.8 µg/mL. Molecular docking suggested that compound 4b and diflumetorim may have similar interactions with complex I NADH oxidoreductase. Density functional theory calculation and pesticide-likeness analysis studies gave a rational explanation of their fungicidal activity. These results indicated that compounds 4b and 4d deserved further optimization according to the principle of pesticide-likeness.

3D-QSAR Combination with Molecular Dynamics Simulations to Effectively Design the Active Ryanodine Receptor Agonists against Spodoptera frugiperda.

Computer-aided molecular modeling was applied to design a series of Spodoptera frugiperda RyR agonists. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to generate 3D-QSAR models. MD simulations in the complex with S. frugiperda native, mutant RyR, and mammalian RyR1 under physiological conditions were used to validate the detailed binding mechanism. Binding free energy calculation by molecular mechanics generalized surface area (MM-GBSA) explained the role of key amino acid residues in ligand-receptor binding. Therefore, 14 new compounds were effectively designed and synthesized, and a bioassay indicated that compounds A-2 and A-3 showed comparable activity to that of chloranthraniliprole with LC50 values of 0.27, 0.18, and 0.20 mg L-1, respectively, against S. frugiperda. Most target compounds also displayed good activity against Mythinma separata at 0.1 mg L-1. Molecular docking and MM-GBSA calculations demonstrated that A-3 had a better binding capacity with native and mutant S. frugiperda RyRs.

CoMFA Directed Molecular Design for Significantly Improving Fungicidal Activity of Novel [1,2,4]-Triazolo-[3,4-b][1,3,4]-thiadizoles.

Target based molecular design via the aid of computation is one of the most efficient methods in the discovery of novel pesticides. Here, a combination of the comparative molecular field analysis (CoMFA) and molecular docking was applied for discovery of potent fungicidal [1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazoles. Bioassay results indicated that the synthesized target compounds 3a, 3b, and 3c exhibited good activity against Alternaria solani, Botrytis cinerea, Cercospora arachidicola, Fusarium graminearum, Physalospora piricola, Rhizoctonia solani, and Sclerotinia sclerotiorum with an EC50 value falling between 0.64 and 16.10 µg/mL. Specially, 3c displayed excellent fungicidal activity against C. arachidicola and R. solani, which was 5 times more potent than the lead YZK-C22. The enzymatic inhibition assay and fluorescence quenching analysis with R. solani pyruvate kinase (RsPK) showed a weaker binding affinity between RsPK and 3a, 3b, or 3c. Transcriptomic analyses showed that 3c exerted its fungicidal activity by disrupting steroid biosynthesis and ribosome biogenesis in eukaryotes. These findings support that 3c is a promising fungicide candidate, and a fine modification from a lead may lead to a totally different mode of action.

Design, Synthesis and Fungicidal Activity of Ester Derivatives of 4-(3,4-Dichloroisothiazole) 7-Hydroxy Coumarin.

The development of new fungicides is vital for safeguarding crops and ensuring sustainable agriculture. Building on our previous finding that 4-(3,4-dichloroisothiazole)-7-hydroxy coumarins can be used as fungicidal leads, 44 novel coumarin ester derivatives were designed and synthesized to evaluate whether esterification could enhance their fungicidal activity. In vitro fungicidal bioassays indicated that compound 2ai displayed good activity against Alternaria solani, Botrytis cinereal, Cercospora arachidicola, Physalospora piricola and Sclerotinia sclerotiorum, with an EC50 value ranging from 2.90 to 5.56 µg/mL, comparable to the lead compound 1a, with its EC50 value ranging from 1.92 to 9.37 µg/mL. In vivo bioassays demonstrated that compounds 1a, 2ar and 2bg showed comparable, excellent efficacy against Pseudoperonospora cubensis at a dose of 25 µg/mL. Our research shows that the esterification of 4-(3,4-dichloroisothiazole) 7-hydroxycoumarins results in a fungicidal activity equivalent to that of its lead compounds. Furthermore, our density functional theory (DFT) calculations and 3D-QSAR modeling provide a rational explanation of the structure-activity relationship and offer valuable insights to guide further molecular design.

Design, synthesis and fungicidal evaluation of novel psoralen derivatives containing sulfonohydrazide or acylthiourea moiety.

To search a novel lead structure for antiphytopathogenic fungus agent, a series of novel psoralen derivatives possessing sulfonohydrazide or acylthiourea structure were designed and synthesized, and their fungicidal activity against seven phytopathogens was evaluated. Their structures were confirmed by melting points, 1H NMR, 13C NMR and HRMS, and the typical crystal structure was determined by X-ray diffraction for validation. Preliminary fungicidal activity showed that some of the title compounds exhibited certain-to-high fungicidal activity. Compound I-13 exhibited good fungicidal activity against Botrytis cinerea, Cercospora arachidicola and Physalospora piricola with EC50 values of 12.49, 13.22 and 12.12 µg/mL, respectively. Compounds II-9 and II-15 showed over 90% inhibition against B. cinerea at 50 µg/mL in vitro. In particular, II-9 exhibited significant higher fungicidal activity with a lower EC50 value of 9.09 µg/mL than the positive control YZK-C22 (13.41 µg/mL). Our studies found that sulfonohydrazide or acylthiourea-containing psoralen derivatives were promising fungicide leads deserve for further study.

Tandem Synthesis of 1,2,3-Thiadiazoles with 3,4-Dichloroisothiazoles and Hydrazines under External Oxidant- and Sulfur-Free Conditions.

1,2,3-Thiadiazoles are among the most important heterocyclic motifs, with wide applications in natural products and medicinal chemistry. Herein, we disclose a tandem reaction for the synthesis of structurally diverse 1,2,3-thiadiazoles from 3,4-dichloroisothiazol-5-ketones and hydrazines. This method is characterized by mild external oxidant- and sulter-free reaction conditions, a broad substrate scope, and easy purification.

Novel [1,2,4]-Triazolo[3,4-b]-[1,3,4]thiadizoles as Potent Pyruvate Kinase Inhibitors for Fungal Control.

To discover novel target-based fungicidal candidates, a molecular design model was established with a three-dimensional (3D) structure of Rhizoctonia solani pyruvate kinase (RsPK) simulated with the AlphaFold 2 and YZK-C22 as a fungicidal lead. A series of novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were rationally designed, synthesized, evaluated for their fungicidal performance, and validated for their mode of action. The in vitro bioassays with R. solani indicated that compounds 5g, 5o, and 5z with an EC50 value ranging from 1.01 to 1.54 µg/mL displayed higher fungicidal activity than the positive control YZK-C22 with its EC50 of 3.14 µg/mL. Especially, 5o exhibited high potency and a broad spectrum against Alternaria solani, Botrytis cinerea, Cercospora arachidicola, Physalospora piricola, R. solani, and Sclerotinia sclerotiorum with its EC50 value falling between 1.54 and 13.10 µg/mL. Like all positive controls, 5g, 5o, and 5z showed excellent in vivo growth inhibition against Pseudoperonospora cubensis at 200 µg/mL. Even though the PK enzymatic inhibition assay showed that 5o was approximately 2.6 times less active than YZK-C22 (IC50: 29.14 vs 11.15 µg/mL, respectively), the similar fluorescence quenching patterns of RsPK by 5o and YZK-C22, and the docking results of interactions between RsPK and 5o or YZK-C22 implied that they might share the similar binding site in the RsPK active pocket. Our studies suggested that 5o could be used as a potent fungicidal lead for further optimization. The results of comparative molecular field analysis (CoMFA) provided a direction for further molecular design.

Design, Synthesis, and Evaluation of Novel 4-Chloropyrazole-Based Pyridines as Potent Fungicide Candidates.

A rational molecular design approach was developed in our laboratory to guide the discovery of novel sterol biosynthesis inhibitors. Based on the application of bioactivities of heterocyclic rings and molecular docking targeting the sterol biosynthesis 14α-demethylase, a series of 4-chloropyrazole-based pyridine derivatives were rationally designed, synthesized, and characterized and their fungicidal activities were also evaluated. Bioassay results showed that 7e, 7f, and 7m exhibited commendable, diverse antifungal actions that are comparable to those of the positive controls imazalil and triadimefon. The active compounds' mode of action was further studied by microscopy observations, Q-PCR, and enzyme inhibition assay and discovered that target compounds affect fungal sterol biosynthesis via disturbing RcCYP51 enzyme system. These findings support that their fungicidal mode of action still targets the cytochrome P450-dependent 14α-demethylase as the molecular design did at first. The above results strongly suggest that our rational molecular design protocol is not only practical but also efficient.

Methoxyacrylate Fungicide Candidate CL-15C Also Functions as a Plant Elicitor in Arabidopsis thaliana and Oryza sativa L.

Unlike traditional fungicides targeting fungi, plant elicitors usually lack direct fungicidal activity but improve the plant immune system to resist fungi infection, which has gained increasing attention for better fungi resistance management and environment protection. (E)-methyl-2-(2-((((Z)-(amino-(3,4-dichloroisothiazol-5-yl)methylene)amino)oxy)methyl)phenyl)-2-(methoxyimino)acetate (CL-15C) was found to be a fungicide candidate with a broad spectrum. Here, we studied its immune-inducing ability and mechanism to strengthen the resistance of Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Oryza sativa L. against Magnaporthe oryzae. CL-15C promoted a 2.20- and 1.47-fold increase in phenylalanine ammonia-lyase (PAL) activity in A. thaliana and O. sativa, respectively. It also facilitated a 1.89- and 1.32-fold increase in accumulation of salicylic acid (SA) in A. thaliana and O. sativa, respectively. Differential genes were clustered in the SA signaling pathway at 24 h after a CL-15C treatment in A. thaliana. Because PAL is a rate-limiting enzyme in the phenylalanine metabolic pathway, after a CL-15C treatment, a pal1(PAL 1) mutant was more susceptible to Pst DC3000 when compared with the wild type. Bacterial counts in leaves after a CL-15C treatment showed a 1.11-fold reduction in the pal1 mutant and a 1.54-fold reduction in the wild type. The effect of CL-15C on the PAL enzyme activity and SA content was attenuated in the pal1 mutant. Present experimental data implied that the immune-inducing activity of CL-15C was dependent on PAL gene-mediated synthesis of SA.

Design, Synthesis, and Biological Evaluation of Novel Psoralen-Based 1,3,4-Oxadiazoles as Potent Fungicide Candidates Targeting Pyruvate Kinase.

Pyruvate kinase (PK) has been considered as a promising fungicide target discovered in our previous studies. Natural compounds are important sources for discovery and development of new pesticides. To continue our ongoing studies on the discovery of novel PK-targeted fungicides, a series of novel psoralen derivatives including a 1,3,4-oxadiazole moiety were designed by a computer-aided pesticide molecular design method, synthesized, and evaluated for their fungicidal activity. The bioassay results indicated that compounds 11d, 11e, 11g, 11i, and 12a showed excellent in vitro fungicidal activity against Botrytis cinerea with EC50 values of 4.8, 3.3, 6.3, 5.4, and 3.9 µg/mL, respectively. They were more active than the corresponding positive control YZK-C22 [3-(4-methyl-1,2,3-thiadiazol-5-yl)-6-(trichloromethyl)-[1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazole] (with an EC50 value of 13.4 µg/mL). Compounds 11g and 11i displayed promising in vivo fungicidal activity against B. cinerea with 80 and 70% inhibition at a concentration of 200 µg/mL, respectively. They possessed much higher fungicidal activity than the positive control psoralen and comparable activity with the positive control pyrisoxazole. Enzymatic assays indicated that 11i showed good BcPK inhibition with an IC50 value of 39.6 µmol/L, comparable to the positive control YZK-C22 (32.4 µmol/L). Molecular docking provided a possible binding mode of 11i in the BcPK active site. Our studies suggested that the psoralen-based 1,3,4-oxadiazole 11i could be used as a new fungicidal lead targeting PK for further structural optimization.

Discovery of Novel Triazolothiadiazines as Fungicidal Leads Targeting Pyruvate Kinase.

Pyruvate kinase (PK) was discovered as a potent new target for novel fungicide development. A series of novel triazolothiadiazine derivatives were rationally designed and synthesized by a ring expansion strategy and computer-aided pesticide design using the 3D structure of Rhizoctonia solani PK (RsPK) obtained by homology modeling as a receptor and our previously discovered lead YZK-C22 as a ligand. The in vitro bioassay results indicated that compounds 4g, 6h, 6m, 6n, 6o, and 6p exhibited good activity against R. solani with the EC50 values falling between 10.99 and 72.76 µM. Especially, 6m showed similar potency to YZK-C22 (10.99 vs 11.97 µM of the EC50 value, respectively). The in vivo bioassay results suggested that 6m against R. solani at a concentration of 200 µg/mL displayed a numerically higher inhibition than YZK-C22 (70 vs 60%, respectively). A field experiment validated that 6m at an application rate of 120 g ai/ha showed comparable efficacy against R. solani to thifluzamide at an application rate of 80 g ai/ha (77.80 vs 84.5%, respectively). Enzymatic inhibition suggested that the potency of 6m was about twofold lower than that of YZK-C22 (67.30 vs 32.64 µM of IC50, respectively). Fluorescence quenching studies validated that RsPK was quenched by both 6m and YZK-C22, implying that they both might act at the same target site of PK. A possible binding conformation of 6m in the RsPK active site was depicted by molecular docking. Our studies suggest that 6m could be a fungicidal lead targeting PK.

Design, synthesis and fungicidal activity of 3,4-dichloroisothiazolocoumarin-containing strobilurins.

Twenty-one novel 3,4-dichloroisothiazolocoumarin-containing strobilurins were rationally designed and synthesized. Preliminary bioassay showed that compounds 7c, 7h and 7l exhibited over 80% inhibitory rate against Sclerotinia sclerotiorum at 50 µg/mL, 7c exhibited good activity against S. sclerotiorum with median effective concentration (EC50) of 4.08 µg/mL, while the positive control coumoxystrobin showed EC50 of 1.00 µg/mL. In addition, 7d showed better fungicidal activity with a lower EC50 value of 7.65 µg/mL against Botrytis cinerea than that of positive control trifloxystrobin with its EC50 value of 21.96 µg/mL. This study indicated that 3,4-dichloroisothiazolocoumarin-containing strobilurin was a promising fungicide lead deserved for further study.

Synthesis of novel N-(2-phenyl-3-pyridyl) thiadiazole/isothiazole carboxamide analogs as potent plant elicitors.

BACKGROUND: Plant elicitors are a class of plant protection agents that can stimulate plant immunity against phytopathogen without a potential resistance problem. In searching for novel plant elicitor candidates, a series of novel N-(2-phenyl-3-pyridyl) thiadiazole/isothiazole carboxamide analogs were designed and synthesized. RESULTS: In vitro bioassay showed that all new compounds exhibited weak direct fungicidal activity. However, compounds 3b, 3g, 3n and 3o showed broad spectrum of in vivo activity against four plant fungi tested. In particularly, 3g showed 80% activity against Rhizoctonia solani in a glasshouse at a concentration of 1 µg mL-1 . For induction activity of tobacco against tobacco mosaic virus (TMV), compounds 3c and 3v showed 67% and 68% inhibitory activity, respectively, which were superior to the positive controls ribavirin and ningnanmycin. Compound 3g showed moderate induction activity (41%). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis found that, 3g could up-regulate expression of genes that are related to reactive oxygen species (ROS), pathogenesis-related protein (PRP) and salicylic acid (SA) signalling. CONCLUSION: These results indicated that 3g as an elicitor candidate might act on the SA signalling pathway. According to our findings, N-(2-phenyl-3-pyridyl) thiadiazole/isothiazole carboxamide analogs might be promising lead scaffolds as a novel plant elicitor for further investigation.

Design, Synthesis, and Evaluation of Novel Isothiazole-Purines as a Pyruvate Kinase-Based Fungicidal Lead Compound.

Target identification is one of the most important bases for novel pesticide development; pyruvate kinase (PK) was discovered as a potent fungicide target in our previous studies. To continue the PK-based fungicide development, novel isothiazole-purine derivatives were rationally designed and synthesized. Bioassay results showed that compound 5ai displayed excellent in vitro activity against Rhizoctonia solani with an EC50 of 1.5 µg/mL, which was superior to those of positive controls diflumetorim with its EC50 of 19.8 µg/mL and PK-based lead YZK-C22 with its EC50 of 4.2 µg/mL. Compounds 3b (5.2 µg/mL) and 3c (4.5 µg/mL) displayed better activities against Gibberella zeae with their EC50s falling between 4.0 and 5.5 µg/mL, while YZK-C22 showed an EC50 of 6.4 µg/mL. In addition, 5ah exhibited promising in vivo activity against Erysiphe graminis and Puccinia sorghi Schw. with 100% efficacy at 10 µg/mL and 90% efficacy at 2 µg/mL against P. sorghi Schw. Compound 5ai showed good PK inhibitory activity with an IC50 of 38.8 µmol/L, and it was well docked into the active site of the target enzyme PK, which was slightly more active than YZK-C22 with its IC50 of 42.4 µmol/L. Our studies discovered that isothiazole-purines were PK-based fungicidal leads deserving of further study.

Discovery of Novel 3,4-Dichloroisothiazole-Containing Coumarins as Fungicidal Leads.

Natural products are one of the resources for discovering novel fungicidal leads. As a natural fungicide, osthole was used as a coumarin-based lead compound for the development of novel fungicides. Here, a series of 3,4-dichloroisothiazole-containing 7-hydroxycoumarins were rationally designed, synthesized, and characterized by introducing a bioactive substructure, 3,4-dichloroisothiazole, into the coumarin skeleton. In vitro bioassay indicated that compound 7g displayed good activity against Rhizoctonia solani, Physalospora piricola, Sclerotinia sclerotiorum, and Botrytis cinerea. Its median effective concentration (EC50) value against each of these fungi fell between 0.88 and 2.50 µg/mL, which was much lower than that of osthole against the corresponding pathogen (between 7.38 and 74.59 µg/mL). In vivo screening validated that 7k exhibited 100%, 60%, and 20% efficacy against R. solani Kühn at 200, 100, and 50 µg/mL, respectively. RNA sequence analysis implied that growth inhibition of R. solani by 7k might result from potential disruptions of fungal membrane formation and intracellular metabolism. Furthermore, a field experiment with cucumber plants indicated that 7b showed 62.73% and 74.03% efficacy against Pseudoperonospora cubensis (Berk. & Curt.) Rostov. at rates of 12.5 g a.i./ha and 25 g a.i./ha, respectively, which showed no significant difference between 7b and osthole at 30 g a.i./ha. Our studies suggested that 7b, 7g, and 7k might be used as fungicidal leads for further optimization.