Design, synthesis and antifungal evaluation of novel indoline‐containing pyrazole‐4‐carboxamide derivatives as potential succinate dehydrogenase inhibitors

Abstract

BACKGROUND

Plant diseases caused by various pathogenic fungi significantly reduce productivity and threaten food safety. Chemical fungicides remain a cornerstone in the management of fungal infections afflicting crops. In search for more potent succinate dehydrogenase inhibitors (SDHIs) for crop protection, a series of novel indoline‐containing pyrazole‐4‐carboxamide derivatives were designed and synthesized.

RESULTS

The assay results indicated that the majority of compounds incorporating a benzyl group displayed remarkable antifungal efficacies. Notably, compound 6j emerged as a standout, exhibiting the highest potency against Rhizoctonia solani, with a half‐maximal effective concentration (EC₅₀) of 0.04 μg mL⁻¹, surpassing Boscalid (EC₅₀ = 0.40 μg mL⁻¹) and Fluxapyroxad (EC₅₀ = 0.06 μg mL⁻¹). Furthermore, compound 6i showed potent antifungal effects against Sclerotinia sclerotiorum and Valsa mali, with EC₅₀ values of 1.79 μg mL⁻¹ and 3.54 μg mL⁻¹, respectively. In vivo tests confirmed that compound 6j effectively inhibited the growth of R. solani on rice leaves, while compound 6i suppressed S. sclerotiorum on oilseed rape and V. mali on apple fruits. Further antifungal mechanism studies revealed that compound 6j shared a similar mode of action with Fluxapyroxad.

CONCLUSION

These findings indicated that compounds 6j and 6i could serve as promising SDHI candidates for crop protection, warranting further investigation. © 2026 Society of Chemical Industry.

This study demonstrated that compounds 6j and 6i exhibited good antifungal activities. A mechanism study indicated that compound 6j might be a potential succinate dehydrogenase inhibitor (SDHI). These results suggested that compounds 6j and 6i could serve as promising SDHI candidates for crop protection.

1. INTRODUCTION

Plant diseases caused by diverse plant pathogenic fungi not only lead to significant reductions in crop productivity but also pose serious threats to food safety. ¹ In addition, mycotoxins produced by these fungal pathogens represent a substantial health risk to both humans and livestock, potentially causing a wide spectrum of acute and chronic health issues, including immunosuppression, organ damage, and carcinogenic effects. ² Consequently, effective management of fungal diseases is essential for ensuring food security, maintaining agricultural sustainability, and protecting public health. Currently, chemical fungicides remain the primary strategy for controlling fungal infections in crops. ³ , ⁴ Among the various classes of fungicides, succinate dehydrogenase inhibitors (SDHIs) have emerged as particularly effective due to their potent antifungal activities and unique mode of action, which distinguishes them from earlier generations of fungicides such as triazoles, benzimidazoles, and strobilurins. ⁵ , ⁶ , ⁷ SDHIs specifically target succinate dehydrogenase (SDH), a key enzyme in the tricarboxylic acid (TCA) cycle, which is essential for cellular respiration and energy production in fungal cells. By blocking electron transfer within mitochondria, SDHIs disrupt fungal metabolism, impair adenosine triphosphate (ATP) generation, and ultimately inhibit fungal growth and proliferation. ⁸ , ⁹ To date, 25 carboxamide‐based SDHI fungicides with outstanding antifungal properties have been commercialized and are widely employed to protect crops such as cereals, fruits, vegetables, and ornamental plants from a broad spectrum of fungal pathogens. ¹⁰ However, the extensive and sometimes improper use of SDHIs has raised concerns regarding the development of resistance. The single‐site mode of action of these fungicides, when combined with prolonged exposure due to overuse or misuse, has accelerated the emergence of resistant fungal strains. ¹¹ , ¹² , ¹³ , ¹⁴ Such resistant populations threaten the long‐term efficacy of SDHI fungicides, potentially leading to reduced disease control, decreased crop yields, and higher economic costs associated with alternative treatments. The proliferation of resistant pathogens represents not only an agricultural problem but also a critical issue for global food security, as it may compromise food quality, availability, and affordability. ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ Given these challenges, there is an urgent need to develop novel SDHI fungicides with improved resistance profiles, broader spectrum activity, and enhanced environmental compatibility.

The chemical structure of SDHI fungicides can generally be divided into three distinct components: an acid moiety, a lipophilic amine portion and an amide function in the central core. ⁵ These structural elements collectively determine the fungicide's biological activity, with the acid core typically responsible for the interaction with the target enzyme, the hydrophobic tail influencing membrane permeability and pharmacokinetic properties, and the amide bond facilitating efficient binding to the active site of the enzyme. ¹⁰ Interestingly, approximately half of commercially available SDHI fungicides contain a pyrazole‐4‐carboxamide fragment, underscoring its prominence as the most common structural motif in this class of fungicides (Fig. 1(A)). ¹⁹ This widespread occurrence highlighted the significance of the pyrazole‐4‐carboxamide structure in conferring antifungal efficacy. Numerous studies have demonstrated that the introduction of a novel hydrophobic tail into the pyrazole‐4‐carboxamide scaffold is one of the most effective strategies for the development of SDHI fungicides. ²⁰ , ²¹ , ²² , ²³

Figure 1.

(A) Structures of commercialized pyrazole‐4‐carboxamides as SDHIs. (B) Design strategy of target compounds.

Indoline skeletons are prevalent in a wide array of natural products and bioactive molecules, representing one of the most fundamental and versatile classes of nitrogen‐containing heterocycles. ²⁴ These structures are well known for their diverse and potent biological activities, including anticancer, ²⁵ antimalarial, ²⁶ , ²⁷ antifungal, ²⁸ antiviral, ²⁹ and herbicidal effects. ³⁰ Given the unique physicochemical and pharmacophoric properties of the indoline scaffold, such as its ability to enhance molecular rigidity, hydrophobicity, and hydrogen‐bonding potential, we sought to incorporate the indoline scaffold into pyrazole‐4‐carboxamides as the hydrophobic tail of SDHIs through a molecular hybridization strategy (Fig. 1(B)). This rational design approach facilitated the efficient synthesis of three series of pyrazole‐4‐carboxamide derivatives with benzyl (6a–6o), acyl (9a–9h), and sulfonyl (12a–12g) substituents, using a straightforward synthetic route (Fig. 2). The newly synthesized compounds were systematically evaluated for their in vitro antifungal activities against five major plant pathogenic fungi, providing a comprehensive assessment of their biological potential. Detailed structure–activity relationships (SARs) were discussed and summarized. Several promising lead compounds demonstrating superior in vitro antifungal activities were further selected for in vivo antifungal evaluation to assess their practical efficacy. Furthermore, scanning electron microscopy (SEM), SDH enzymatic inhibition assays, molecular docking studies, Molecular dynamics (MD) simulations, density functional theory (DFT) calculations, and electrostatic potential (ESP) mapping were employed to gain a deeper mechanistic understanding of their antifungal mechanism.

Figure 2.

Synthesis of target compounds.

2. MATERIALS AND METHODS

2.1. Chemicals and instruments

All chemicals for this study were sourced from Aladdin in Shanghai, China, guaranteeing high purity and consistency. The instrumentation and analytical equipment used were detailed in our previous work ³¹ and available in the Supporting Information Method S1.

2.2. Synthetic procedures

2.2.1. General synthesis procedure for intermediate 2

The synthetic procedures for intermediate 2 were conducted using the methodology outlined in our previous research. ³¹

2.2.2. General synthesis procedure for intermediates 4a–4o

In a 100 mL round‐bottom flask, commercial compound 3 (4.0 mmol), potassium carbonate (K₂CO₃, 12.0 mmol), and substituted benzyl bromide (8.0 mmol) were mixed in 50 mL of acetonitrile (CH₃CN) and stirred at reflux. After the reaction, 50 mL of water was added, and the mixture was extracted with dichloromethane (DCM). The organic layers were collected, dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by flash column chromatography using a petroleum ether and ethyl acetate mixture (40:1), yielding intermediates 4a–4o.

2.2.3. General synthesis procedure for intermediates 7a–7h and 10a–10g

A solution of compound 3 (3.0 mmol) and either substituted acyl chloride or substituted benzene sulfonyl chloride (4.5 mmol) was prepared in 25 mL of DCM. Triethylamine (Et₃N, 9.0 mmol) was added at room temperature, and the mixture was stirred overnight. Afterward, 15 mL of water was added, and the mixture was extracted with DCM. The organic layers were dried, concentrated, and purified by flash column chromatography using a petroleum ether and ethyl acetate mixture (10:1), yielding intermediates 7a–7h and 10a–10f.

2.2.4. General synthesis procedure for intermediates 5a–5o, 8a–8h, and 11a–11g

A mixture of intermediates 4a–4o, 7a–7h or 10a–10g (2.0 mmol) and stannous chloride dihydrate (10.0 mmol) was dissolved in 30 mL of anhydrous ethanol (EtOH) and heated to reflux overnight. After cooling, excess EtOH was removed using a rotary evaporator, and the residue was extracted with DCM. The organic layers were combined, dried over anhydrous Na₂SO₄, and concentrated, yielding intermediates 5a–5o, 8a–8h, and 11a–11g without further purification.

2.2.5. General synthesis procedure for target compounds 6a–6o, 9a–9h, and 12a–12g

A solution of intermediates 5a–5o, 8a–8h or 11a–11g (1.0 mmol) and intermediate 2 (1.5 mmol) was prepared in 15 mL of DCM. Et₃N (3.0 mmol) was added at room temperature, and the mixture was stirred overnight. Afterward, 15 mL of water was added, and the mixture was extracted with DCM. The organic layers were dried, concentrated, and purified by flash column chromatography using a petroleum ether and ethyl acetate mixture (4:1), yielding target compounds 6a–6o, 9a–9h, and 12a–12g. Detailed physical properties of these compounds are provided in Supporting Information Data S1.

2.3. In vitro antifungal activities

The widely recognized mycelia growth inhibition method ³² , ³³ was employed to assess the in vitro antifungal efficacies of the target compounds against five fungal species: Rhizoctonia solani, Sclerotinia sclerotiorum, Valsa mali, Fusarium graminearum, and Botrytis cinerea. The detailed experimental procedures and methodologies are available in Method S2.

2.4. In vivo antifungal activities

Following literature methods and practical adjustments, the protective and curative activities of compound 6j against R. solani on rice leaves and compound 6i against S. sclerotiorum on oilseed rape leaves and V. mali on apple fruit were evaluated. ³⁴ , ³⁵ , ³⁶ Detailed information is provided in Method S3.

2.5. In vitro SDH inhibition assay

The activities of the SDH enzyme for compound 6j, which demonstrated significant antifungal efficacy against R. solani, were assessed using a SDH assay kit (BC0955; Solebro, College of Tea and Food Science, Xinyang Normal University; Address: Xinyang 464000, Henan Province, China). This evaluation was carried out in accordance with established methodologies outlined in the scientific literature. ³⁷ , ³⁸ Comprehensive procedural details are available in Method S4.

2.6. SEM analysis

The SEM analysis was conducted following established protocols from the literature. ³⁹ , ⁴⁰ Briefly, 5 mm mycelial discs were excised from actively growing cultures of R. solani and placed on potato dextrose agar (PDA) plates. Two experimental groups were prepared: one served as a negative control without additional compounds, while the other was supplemented with 1 μg mL⁻¹ of compound 6j and controls. The plates were incubated at 27 °C to promote optimal fungal growth and interaction with the test compounds. After incubation, the effects of the compounds on R. solani mycelium morphology were analyzed using SEM.

2.7. Molecular docking and MD simulations

To better understand the interaction modes and binding mechanisms of the target compounds with the SDH protein, molecular docking and MD simulations were performed. The docking simulations were executed using Autodock 4.2.6, utilizing the crystal structure of SDH retrieved from the protein database (PDB entry: 4YTP). ⁴¹ MD simulations were conducted with Gromacs 2022.3 software. ⁴² A detailed description of the methodologies for both simulations can be found in Methods S5 and S6.

2.8. DFT and ESP calculation

The DFT and ESP calculations for compound 6j and Fluxapyroxad were conducted using their optimized structures within the Gaussian 16 computational chemistry program. These calculations employed the B3LYP/6‐311G** theory level, which integrates the Becke three‐parameter hybrid functional (B3LYP) with a triple‐zeta basis set enhanced by diffuse and polarization functions (6‐311G**). ⁴³

3. RESULTS AND DISCUSSION

3.1. Synthesis of target compounds

The synthetic pathway for the target compounds is illustrated in Fig. 2. Proton (¹H) nuclear magnetic resonance (NMR), carbon‐13 (¹³C) NMR, and high‐resolution mass spectrometry (HRMS) were employed to confirm the chemical structures of these synthesized compounds. Detailed data and results from all analytical methods are provided in Data S2.

3.2. Antifungal activity and SAR discussion

The preliminary fungicidal activities of all target compounds were assessed in vitro against five different fungal strains at a concentration of 20 μg mL⁻¹. Boscalid and Fluxapyroxad served as positive controls to evaluate the efficacies of the newly synthesized compounds. The results are summarized in Table 1.

Table 1.

In vitro antifungal effects (%) of all target compounds at 20 μg mL^{−1 a}

Compound	R. s. b	S. s. c	V. m. d	F. g. e	B. c. f
6a	86.6 ± 1.4	82.1 ± 1.2	58.5 ± 2.1	51.7 ± 2.6	35.4 ± 0.9
6b	73.3 ± 2.4	50.6 ± 0.9	29.7 ± 1.6	38.3 ± 0.3	34.0 ± 2.9
6c	83.9 ± 2.1	86.4 ± 1.5	29.2 ± 2.3	47.2 ± 1.1	22.9 ± 1.9
6d	82.5 ± 1.1	84.7 ± 1.9	16.4 ± 1.6	35.0 ± 1.6	30.7 ± 0.9
6e	79.6 ± 1.8	83.3 ± 1.8	23.7 ± 1.0	52.8 ± 1.3	33.4 ± 1.8
6f	77.8 ± 1.3	78.3 ± 1.8	44.6 ± 2.4	51.7 ± 1.6	31.5 ± 0.9
6g	75.3 ± 0.8	68.6 ± 0.9	35.4 ± 0.4	46.7 ± 2.1	32.6 ± 1.2
6h	74.0 ± 1.3	42.0 ± 1.8	48.2 ± 1.6	51.1 ± 1.9	39.4 ± 2.5
6i	90.6 ± 0.8	89.5 ± 0.7	90.9 ± 0.7	65.0 ± 1.5	46.5 ± 0.6
6j	96.8 ± 1.7	88.3 ± 0.6	85.2 ± 1.6	61.7 ± 1.1	38.2 ± 1.1
6k	86.0 ± 1.6	83.3 ± 1.5	81.2 ± 2.4	61.7 ± 1.5	52.8 ± 0.5
6l	88.5 ± 1.0	79.8 ± 1.2	80.1 ± 1.0	64.4 ± 1.9	34.6 ± 2.8
6m	78.5 ± 2.1	71.2 ± 0.5	72.7 ± 1.5	63.9 ± 0.9	40.3 ± 2.2
6n	81.5 ± 1.5	77.5 ± 0.9	80.8 ± 1.8	62.8 ± 1.1	48.0 ± 0.9
6o	72.7 ± 0.7	34.0 ± 0.6	28.2 ± 2.1	42.8 ± 1.9	23.0 ± 0.4
9a	70.9 ± 1.3	40.7 ± 2.2	30.8 ± 0.5	36.7 ± 1.6	26.7 ± 2.5
9b	18.5 ± 1.4	15.0 ± 2.6	15.4 ± 1.9	22.8 ± 2.3	18.8 ± 1.2
9c	43.8 ± 1.0	21.3 ± 1.1	25.6 ± 1.1	39.4 ± 1.9	33.9 ± 1.9
9d	71.1 ± 2.2	33.3 ± 2.7	41.5 ± 1.4	51.7 ± 2.5	47.3 ± 1.6
9e	46.8 ± 1.9	41.3 ± 0.7	48.7 ± 2.1	58.3 ± 1.7	52.7 ± 1.4
9f	21.4 ± 1.1	29.4 ± 1.1	24.6 ± 0.5	33.3 ± 3.0	38.9 ± 0.8
9g	15.4 ± 1.6	43.9 ± 0.8	18.0 ± 1.3	23.9 ± 1.7	21.5 ± 2.1
9h	57.5 ± 1.9	26.7 ± 1.8	50.8 ± 1.8	41.7 ± 1.8	34.2 ± 1.9
12a	70.8 ± 0.7	71.7 ± 0.6	70.1 ± 1.4	50.0 ± 3.3	22.4 ± 0.9
12b	58.8 ± 1.9	30.7 ± 0.8	56.4 ± 0.8	45.6 ± 0.4	19.4 ± 1.9
12c	62.5 ± 1.2	28.7 ± 1.5	38.0 ± 2.2	42.8 ± 1.9	23.0 ± 0.9
12d	72.1 ± 1.7	73.3 ± 1.0	71.6 ± 2.1	45.6 ± 2.6	46.7 ± 0.9
12e	56.7 ± 0.7	24.7 ± 0.5	54.6 ± 0.8	47.8 ± 1.9	36.4 ± 1.6
12f	52.5 ± 1.1	26.7 ± 1.0	41.5 ± 1.4	37.8 ± 3.4	26.1 ± 1.8
12g	52.9 ± 1.3	20.7 ± 0.9	32.8 ± 1.6	44.4 ± 2.5	30.3 ± 1.9
Boscalid	86.2 ± 1.2	97.4 ± 1.5	37.6 ± 1.1	56.7 ± 1.6	66.7 ± 1.9
Fluxapyroxad	96.5 ± 1.7	98.8 ± 0.7	49.8 ± 1.4	58.6 ± 1.3	79.2 ± 0.9

^a The data are the mean values of triplicate experiments.

^b Rhizoctonia solani.

^c Sclerotinia sclerotiorum.

^d Valsa mali.

^e Fusarium graminearum.

^f Botrytis cinerea.

Based on the data presented in Table 1, the target compounds featuring a benzyl group on the indoline scaffold (6a–6o) consistently demonstrated significantly superior antifungal activities compared to their acyl‐substituted (9a–9h) and sulfonyl‐substituted (12a–12g) counterparts. This trend strongly suggested that the presence of a benzyl group enhances the compound's efficacy against the tested fungal strains, likely due to its unique combination of electronic effects and steric properties, which may facilitate stronger interactions with fungal target sites and improve membrane permeability. In contrast, compounds bearing acyl or sulfonyl groups generally exhibited weaker antifungal performance, highlighting the critical role of functional group selection in optimizing bioactivity within this class of derivatives. For R. solani, all benzyl‐containing compounds displayed excellent antifungal activity, with inhibition rates exceeding 70% at a concentration of 20 μg mL⁻¹. Remarkably, compounds 6i and 6j stood out, demonstrating inhibition rates surpassing 90%, which not only outperformed Boscalid (86.1%) but also approached the high efficacy of Fluxapyroxad (96.5%). Conversely, among the acyl‐ and sulfonyl‐substituted derivatives, only compounds 9a, 9d, 12a, and 12d achieved inhibition rates over 70%, underscoring the relative inferiority of these functional groups in conferring potent antifungal activity against R. solani. Regarding S. sclerotiorum, most benzyl‐containing compounds exhibited significant fungicidal effects, with inhibition rates above 70%, emphasizing their strong potential against this pathogen. In particular, compound 6i emerged as the most promising candidate, achieving an inhibition rate of 89.5%. Although slightly lower than Boscalid (97.4%) and Fluxapyroxad (98.8%), this value demonstrated potent antifungal efficacy and indicated practical utility for controlling S. sclerotiorum in agricultural settings. By comparison, only two sulfonyl‐bearing compounds (12a and 12d) showed inhibition rates above 70%, while none of the acyl‐substituted derivatives displayed notable activity against this fungus. In the case of V. mali, eight compounds demonstrated substantial antifungal activity, with inhibition rates exceeding 70%, while the remaining compounds were largely ineffective. Among these, compound 6i again showed the most promising activity, with an inhibition rate of 90.9%, far surpassing Boscalid (37.6%) and Fluxapyroxad (49.8%), indicating its potential as a highly effective candidate for managing this pathogen. However, as for F. graminearum and B. cinerea, none of the tested compounds achieved strong antifungal activity, with inhibition rates remaining below 70%. This result suggested that the spectrum of action for these derivatives is somewhat restricted, highlighting the challenge of developing universally potent antifungal agents within this series. Notably, compounds 6i and 6j demonstrated broader antifungal spectra compared to the other derivatives, combining high potency against multiple pathogens with promising inhibition rates. These features positioned them as particularly attractive candidates for the development of versatile and efficient agricultural fungicides, a key consideration for practical field applications where multiple fungal threats may be present simultaneously.

To further assess the fungicidal potential and elucidate the SARs of the target compounds, only those exhibiting exceptional antifungal activities (inhibition rates > 70%) were selected for detailed analysis. This selective approach enabled a focused examination of the most promising candidates, providing clearer insights into the chemical features that contribute to potent antifungal effects. By concentrating on high‐performing compounds, subtle differences in molecular structure and their impact on bioactivity could be more accurately discerned. The half‐maximal effective concentration (EC₅₀) values of these selected compounds against R. solani are summarized in Table 2 and corresponding EC₅₀ values for S. sclerotiorum and V. mali are provided in Table 3.

Table 2.

Half‐maximal effective concentration (EC₅₀) values (μg mL⁻¹) of selected target compounds against Rhizoctonia solani

Compound	Regression equation	R 2	EC50 (95% confidence interval)
6a	y = 5.455x + 0.597	0.991	0.17 (0.07–0.42)
6b	y = 0.655x + 4.965	0.998	1.13 (0.51–2.53)
6c	y = 0.703x + 5.287	0.991	0.40 (0.19–0.81)
6d	y = 0.666x + 5.257	0.978	0.41 (0.23–0.75)
6e	y = 0.596x + 5.212	0.988	0.44 (0.22–0.87)
6f	y = 0.643x + 5.130	0.996	0.54 (0.29–1.04)
6g	y = 0.765x + 5.127	0.983	0.68 (0.38–1.22)
6h	y = 4.989x + 0.534	0.981	1.05 (0.39–2.82)
6i	y = 0.507x + 5.515	0.998	0.10 (0.03–0.33)
6j	y = 0.444x + 5.616	0.994	0.04 (0.01–0.20)
6k	y = 0.890x + 5.364	0.992	0.39 (0.25–0.62)
6l	y = 0.599x + 5.491	0.986	0.15 (0.05–0.48)
6m	y = 0.646x + 5.150	0.999	0.59 (0.31–1.13)
6n	y = 5.227x + 0.828	0.999	0.53 (0.32–0.89)
6o	y = 0.535x + 4.928	0.998	1.36 (0.47–3.98)
9a	y = 0.911x + 4.568	0.997	2.98 (0.86–10.31)
9d	y = 0.784x + 4.650	0.988	2.79 (0.72–10.90)
12a	y = 0.547x + 4.799	0.994	2.34 (0.47–11.67)
12d	y = 0.297x + 4.959	0.975	1.37 (0.12–16.05)
Boscalid	y = 0.462x + 5.184	0.983	0.40 (0.15–1.06)
Fluxapyroxad	y = 0.739x + 5.896	0.980	0.06 (0.03–0.11)

Table 3.

Half‐maximal effective concentration (EC₅₀) values (μg mL⁻¹) of selected compounds against Sclerotinia sclerotiorum and Valsa mali

Fungi	Compound	Regression equation	R 2	EC50 (95% confidence interval)
Sclerotinia sclerotiorum	6a	y = 1.137x + 4.405	0.987	3.34 (2.23–5.00)
	6c	y = 0.649x + 4.729	0.994	2.62 (1.29–5.30)
	6d	y = 0.668x + 4.714	0.994	2.68 (1.35–5.31)
	6e	y = 0.978x + 4.327	0.991	4.88 (3.10–7.69)
	6f	y = 1.011x + 4.268	0.984	5.29 (3.17–8.85)
	6i	y = 0.746x + 4.811	0.979	1.79 (0.86–3.72)
	6j	y = 0.280x + 4.921	0.983	1.92 (0.51–7.31)
	6k	y = 0.727x + 4.714	0.999	2.48 (1.30–4.73)
	6l	y = 0.880x + 4.641	0.999	2.56 (1.38–4.77)
	6m	y = 1.116x + 4.214	0.978	5.06 (3.46–7.39)
	6n	y = 0.863x + 4.626	0.984	2.71 (1.58–4.66)
	12a	y = 1.054x + 4.093	0.992	7.25 (4.21–12.49)
	12d	y = 1.089x + 4.093	0.996	6.81 (4.51–10.29)
	Boscalid	y = 0.752x + 5.257	0.991	0.46 (0.07–2.83)
	Fluxapyroxad	y = 0.461x + 5.662	0.997	0.04 (0.01–0.50)
Valsa mali	6i	y = 1.342x + 4.264	0.998	3.54 (2.51–4.98)
	6j	y = 1.180x + 4.046	0.996	6.43 (4.28–9.68)
	6k	y = 1.428x + 3.784	0.996	7.10 (4.97–10.15)
	6l	y = 1.003x + 4.116	0.991	7.62 (4.62–12.56)
	6m	y = 1.173x + 3.895	0.996	8.75 (5.50–13.90)
	6n	y = 0.877x + 4.180	0.995	8.61 (4.78–15.52)
	12a	y = 1.111x + 3.743	0.990	13.55 (7.56–24.28)
	12d	y = 0.950x + 4.077	0.988	9.38 (5.32–16.52)
	Boscalid			> 20
	Fluxapyroxad			> 20

As shown in Table 2, all benzyl‐containing compounds demonstrated strong antifungal activities against R. solani, while only two compounds with acyl groups and two with sulfonyl groups exhibited similarly potent antifungal effects. Among these, compound 6j emerged as the standout candidate, exhibiting remarkable antifungal potency with an EC₅₀ value of 0.04 μg mL⁻¹. This value was ten times more potent than Boscalid (EC₅₀ = 0.40 μg mL⁻¹) and one and a half times more effective than Fluxapyroxad (EC₅₀ = 0.06 μg mL⁻¹), underscoring its potential as a highly effective fungicide for controlling R. solani. These results strongly suggest that compound 6j could serve as a more powerful alternative for R. solani management, offering superior performance in agricultural applications. In general, compounds containing a benzyl group (6a–6o) exhibited significantly higher antifungal activities than those with acyl (9a–9h) or sulfonyl (12a–12g) groups. Notably, only four compounds (9a, 9d, 12a, and 12d) displayed good antifungal activities against R. solani, with EC₅₀ values of 2.98, 2.79, 2.34, and 1.37 μg mL⁻¹, respectively. While these values were moderate, they suggested that the acyl and sulfonyl groups might not be optimal for achieving high antifungal potencies, possibly due to weaker interactions with fungal target sites or reduced membrane permeability, which could hinder the compound's efficacy. In contrast, the benzyl‐containing compounds consistently exhibited superior antifungal activities, highlighting the critical role of the benzyl group in enhancing bioactivity. A closer examination of the effect of substituents on the benzyl ring revealed that the presence of electron‐donating groups did not significantly improve antifungal activity. In fact, electron‐withdrawing substituents were more effective in boosting the antifungal potential of these compounds. For example, compounds 6b (EC₅₀ = 1.13 μg mL⁻¹) and 6o (EC₅₀ = 1.36 μg mL⁻¹), which contained electron‐donating groups such as a methyl group and a 3,5‐dimethoxy group, exhibited weaker activities compared to those with halogen atoms (fluorine (F), chlorine (Cl), bromine (Br), iodine (I)) in the benzyl position. Among the halogen‐containing compounds, a clear trend emerged in which the antifungal activities increased as the electron‐withdrawing ability of the halogen increased. The compounds 6c (4‐F), 6d (4‐Cl), 6e (4‐Br), and 6f (4‐I) exhibited the following order of potency: compound 6d (EC₅₀ = 0.40 μg mL⁻¹) > compound 6e (EC₅₀ = 0.41 μg mL⁻¹) > compound 6f (EC₅₀ = 0.44 μg mL⁻¹) > compound 6g (EC₅₀ = 0.54 μg mL⁻¹), indicating that halogens were particularly effective in enhancing antifungal efficacy. Moreover, the position of the halogen substituent on the benzyl ring played a crucial role in modulating the antifungal activity of the compounds. Specifically, meta‐substitution on the benzyl group resulted in superior antifungal activity compared to ortho‐substitution, which in turn outperformed para‐substitution. For instance, compound 6j, which contained a 3‐Cl (meta‐substitution) on the benzyl group, exhibited higher antifungal activity (EC₅₀ = 0.04 μg mL⁻¹) than compound 6l (EC₅₀ = 0.15 μg mL⁻¹), which had a 2‐Cl (ortho‐substitution), and compound 6d (EC₅₀ = 0.41 μg mL⁻¹), which featured a 4‐Cl (para‐substitution). This pattern was further corroborated by the fact that compounds 6i (EC₅₀ = 0.10 μg mL⁻¹), which contained a 3‐F, exerted better antifungal activity than compound 6k (EC₅₀ = 0.39 μg mL⁻¹) with a 2‐F and compound 6c (EC₅₀ = 0.40 μg mL⁻¹) with a 4‐F. These findings emphasized the critical role of the halogen substituent's position on the benzyl ring in optimizing antifungal activity. The meta‐position appeared to enhance the electronic interactions and spatial configuration of the molecule, likely improving its binding affinity and overall interaction with fungal targets. This insight offered valuable guidance for the rational design of future antifungal agents with enhanced potency, particularly those aimed at controlling R. solani. By fine‐tuning the electronic properties of the benzyl group and carefully selecting halogen substituents, it might be possible to further optimize the antifungal potential of these compounds for broader agricultural applications.

As illustrated in Table 3, For S. sclerotiorum, most of the benzyl‐containing compounds exhibited notable antifungal activities, whereas only two sulfonyl‐containing compounds demonstrated meaningful effects, underscoring the critical role of the benzyl substituent in modulating antifungal potency. Among these, compound 6i emerged as the most promising candidate, showing good activity with an EC₅₀ value of 1.79 μg mL⁻¹. Although this potency was considerably lower than that of Fluxapyroxad (EC₅₀ = 0.04 μg mL⁻¹), it approached that of Boscalid (EC₅₀ = 0.46 μg mL⁻¹), suggesting that compound 6i possessed a moderate yet biologically meaningful inhibitory effect against S. sclerotiorum. A closer comparison of the benzyl‐substituted compounds revealed a clear trend in activity: compound 6i (EC₅₀ = 1.79 μg mL⁻¹) > compound 6k (EC₅₀ = 2.48 μg mL⁻¹) > compound 6c (EC₅₀ = 2.62 μg mL⁻¹), and compound 6j (EC₅₀ = 1.92 μg mL⁻¹) > compound 6l (EC₅₀ = 2.56 μg mL⁻¹) > compound 6d (EC₅₀ = 2.68 μg mL⁻¹). These results reinforced the observation that meta‐substitution on the benzyl ring conferred superior antifungal activity, followed by ortho‐substitution, with para‐substitution being the least favorable. This positional effect was consistent with the trends observed against R. solani, suggesting that the SAR of the benzyl substituent was conserved across multiple fungal species. Such a consistent pattern provided important guidance for rational molecular modifications aimed at improving antifungal potency. Regarding V. mali, eight compounds demonstrated meaningful antifungal activities, with EC₅₀ values ranging from 3.54 μg mL⁻¹ to 13.55 μg mL⁻¹. While these activities were moderate, they were significantly more effective than Boscalid and Fluxapyroxad, both of which exhibited EC₅₀ values greater than 20 μg mL⁻¹, indicating a clear advantage of these novel compounds. Among them, compound 6i again stood out as the most potent compound, with an EC₅₀ of 3.54 μg mL⁻¹, highlighting its potential application in controlling V. mali. Notably, compounds 6i and 6j demonstrated the ability to inhibit multiple fungal pathogens, suggesting that they might serve as promising candidates for broad‐spectrum fungicides with practical utility in diverse agricultural contexts.

The SARs of the target compounds, illustrated in Fig. 3, were summarized as follows. (i) Benzyl versus acyl or sulfonyl groups: compounds with a benzyl group on the indoline scaffold demonstrated significantly higher antifungal activities compared to those with acyl or sulfonyl groups. The benzyl group's flexibility likely enhances its binding affinity through favorable electronic and steric interactions with fungal targets, improving molecular docking and membrane penetration. In contrast, acyl and sulfonyl groups, being bulkier and less flexible, may hinder effective interactions with the target, resulting in reduced antifungal potency. (ii) Substitution pattern on benzyl group: the position of substituents on the benzyl ring critically affected antifungal efficacies. Specifically, meta‐substitution proved most effective, surpassing ortho‐substitution, while para‐substitution was least effective. This trend highlighted the importance of spatial orientation and electronic effects; meta‐substituents optimized alignment and reduced steric hindrance, enhancing binding to fungal receptors. (iii) Effect of halogen atoms: antifungal activity increased with the electron‐withdrawing ability of halogens. Heavier halogens like Cl, Br, and I enhanced interactions with fungal targets by increasing the electrophilicity of the aromatic ring. This trend emphasized the role of halogen substitution in modulating the reactivity and binding affinity of compounds. In summary, optimizing antifungal agents involved (i) incorporating a benzyl group for improved activity, (ii) favoring meta‐substitution for better steric and electronic advantages, and (iii) selecting appropriate halogens to enhance electron‐withdrawing properties. Understanding these factors could aid in designing more effective and selective antifungal treatments.

Figure 3.

SARs of target compounds containing indoline group.

3.3. In vivo fungicidal activities

To further assess the application prospect of lead compounds, a thorough in vivo assessment was performed to investigate the in vivo protective efficacies and curative efficacies of compound 6j and controls against R. solani on rice leaves, protective efficacies of compound 6i and controls against S. sclerotiorum on oilseed rape leaves and V. mali on apple fruits. As shown in Fig. 4, compound 6j exhibited outstanding antifungal activity, achieving a remarkable protective efficacy of 97.38% and curative efficacy of 90.09% at a concentration of 100 μg mL⁻¹ against R. solani. These results not only outperformed Boscalid (69.33% and 69.89%) and Fluxapyroxad (88.07% and 83.88%) but also highlighted the potent fungicidal properties of compound 6j against this devastating rice pathogen. In the case of S. sclerotiorum on oilseed rape leaves, compound 6i demonstrated a strong protective efficacy of 83.81%, which was comparable to Boscalid (89.95%) but somewhat lower than Fluxapyroxad (98.31%). Although the protective effect of compound 6i against S. sclerotiorum was slightly less potent, it still presented a promising level of protection, suggesting that compound 6i could serve as an effective alternative in specific agricultural environments where Fluxapyroxad or Boscalid might not be ideal choices. Additionally, in the efficacy tests against V. mali on apple fruits, compound 6i stood out with an exceptional protective efficacy of 89.60%, which significantly surpassed both Boscalid (50.92%) and Fluxapyroxad (70.56%). This superior performance against V. mali positioned compound 6i as a highly effective candidate for managing this important fungal pathogen in apple fruits, showcasing its versatility across different plant species and pathogens. Collectively, these results strongly indicated that compounds 6j and 6i held significant potential as advanced fungicides for the control of R. solani, S. sclerotiorum, and V. mali, offering promising alternatives to current commercial fungicides in diverse agricultural settings.

Figure 4.

(A) In vivo protective efficacies and curative efficacies of compound 6j and controls against Rhizoctonia solani. (B) In vivo protective efficacies of compound 6i and controls against Sclerotinia sclerotiorum. (C) In vivo protective efficacies of compound 6i and controls against Valsa mali.

3.4. Enzymatic inhibition activity of SDH

To investigate the potential antifungal mechanism of these novel pyrazole‐4‐carboxamide derivatives, compound 6j, which exhibited remarkable fungicidal activity against R. solani, was selected as a representative candidate for the SDH enzymatic inhibition assay. As summarized in Table 4, compound 6j displayed significant SDH inhibitory activity, with a half‐maximal inhibitory concentration (IC₅₀) value of 0.70 μm, which was notably superior to that of Boscalid (IC₅₀ = 1.45 μm) and close to Fluxapyroxad (IC₅₀ = 0.26 μm). The strong inhibition of SDH by compound 6j suggested that its antifungal efficacy might arise from interference with the fungal mitochondrial electron transport chain, leading to energy depletion and growth suppression of R. solani. These findings indicated that compound 6j not only possessed potent fungicidal activity but also likely exerted its effect through targeted SDH inhibition, highlighting its potential as a novel SDHI fungicide. The combination of high enzymatic inhibitory activity and effective antifungal performance positioned compound 6j as a promising candidate for further development in the control of R. solani and potentially other pathogenic fungi.

Table 4.

Half‐maximal inhibitory concentration (IC₅₀) values (μm) of selected compounds against succinate dehydrogenase (SDH) of Rhizoctonia solani

Compound	Regression equation	R 2	IC50 (95% confidence interval)
6j	y = 1.475x + 5.226	0.984	0.70 (0.54–0.92)
Boscalid	y = 1.335x + 4.785	0.985	1.45 (1.09–1.92)
Fluxapyroxad	y = 1.518x + 5.887	0.991	0.26 (0.21–0.32)

3.5. SEM analysis

To investigate the subtle micromorphological changes induced in R. solani mycelia following treatment with various antifungal compounds, SEM analysis was performed, as depicted in Fig. 5. The untreated R. solani mycelia (negative control) displayed a smooth, tubular, and unbranched structure (Fig. 5(A),(B)), which was characteristic of healthy, undisturbed fungal growth, indicative of normal cellular function and morphology. However, upon treatment with compound 6j (Fig. 5(C),(D)), Boscalid (Fig. 5(E),(F)), and Fluxapyroxad (Fig. 5(G),(H)) at a concentration of 1 μg mL⁻¹, significant morphological alterations were observed in the mycelia. These changes included pronounced wrinkling, entanglement, and shrinkage, which were clear signs of fungal cell wall disruption. Such alterations were typically associated with the action of antifungal agents targeting the structural integrity of the fungal cell wall and membrane. These observations were particularly noteworthy as they highlighted the potent action of these compounds on the mycelia, disrupting their normal growth and morphology. Among the treatments, compound 6j exhibited morphological changes that were remarkably similar to those induced by the well‐established SDHIs Boscalid and Fluxapyroxad. This similarity suggested that compound 6j might share a similar mechanism of action with these compounds, likely targeting the fungal cell wall or other essential cellular structures, thereby inhibiting the normal development of the mycelia. These results provided compelling evidence that compound 6j exhibited strong antifungal activity, with the potential to disrupt the growth and survival of R. solani. The observed micromorphological changes reinforced the idea that compound 6j might be highly effective as a fungicide, providing an efficient and reliable solution for controlling R. solani in agricultural settings. Given its potent ability to compromise the structural integrity of fungal cells, compound 6j held promise as a key candidate for future fungicide development aimed at protecting rice from R. solani.

Figure 5.

SEM of the hyphae of Rhizoctonia solani. (A, B) Negative control; (C, D) compound 6j at 1 μg mL⁻¹; (E, F) Boscalid at 1 μg mL⁻¹; (G, H) Fluxapyroxad at 1 μg mL⁻¹.

3.6. Molecular docking study and MD simulation

To achieve a deeper understanding of the binding mechanisms between the target compounds and the SDH enzyme, lead compound 6j along with controls Fluxapyroxad and Boscalid were selected for molecular docking studies and MD simulations. As illustrated in Fig. 6, all three compounds, compound 6j (Fig. 6(A),(B)), Boscalid (Fig. 6(C),(D)), and Fluxapyroxad (Fig. 6(E),(F)), were found to occupy the same active site pocket of the SDH enzyme. This observation indicated that although these compounds were chemically diverse, their binding modes share fundamental similarities, highlighting their potential as effective inhibitors of SDH. One of the critical insights gained from the docking analysis was the interaction between key amino acid residues, specifically TYR91 and TRP173, and the carbonyl group present in each compound. The strategic positioning of these residues was essential for stabilizing the ligand–receptor interactions. For instance, the carbonyl group of compound 6j was positioned at a distance of 2.0 Å from TYR91 and 3.2 Å from TRP173, suggesting a favorable proximity conducive to hydrogen bond formation. Similar distances were recorded for Boscalid (1.8 Å from TYR91 and 3.0 Å from TRP173) and Fluxapyroxad (2.0 Å from TYR91 and 2.3 Å from TRP173). Such stable hydrogen bonding implied that the binding of these compounds to the SDH enzyme is not only robust but also selective, which was crucial for their efficacy as inhibitors. This understanding laid the groundwork for designing next‐generation SDHIs that aim to maintain high levels of efficacy while also enhancing stability and mitigating resistance.

Figure 6.

Molecular docking modes of compound 6j (A, B), Boscalid (C, D) and Fluxapyroxad (E, F) with SDH (PDB code: 4YTP).

Subsequently, 50 ns MD simulations were conducted on three distinct protein–ligand complexes: SDH–Boscalid, SDH–Fluxapyroxad, and SDH–6j. As illustrated in Fig. 7(A), the average root mean square deviation (RMSD) values for the complexes were 0.39 nm for SDH–6j, 0.29 nm for SDH–Boscalid, and 0.28 nm for SDH–Fluxapyroxad. Notably, all RMSD values remained consistently below 1 nm throughout the entirety of the simulations. These results indicated that the MD trajectories for all three complexes had reached a state of equilibrium and maintained stability over the simulation duration, thereby confirming the reliability of the findings for subsequent analyses. The consistently low RMSD values across the complexes underscored the effectiveness of the ligands (6j, Boscalid, and Fluxapyroxad) in stabilizing the SDH protein structure, ensuring that the protein retained a stable conformation throughout the entire 50 ns simulation period. Following this confirmation of stability, we proceeded to analyze the fluctuations of the amino acid residues within the complexes. The root mean square fluctuation (RMSF) values for all residues were calculated across the 50 ns trajectories, providing valuable insights into the flexibility of individual residues in response to ligand binding. As shown in Fig. 7(B), the SDH–6j complex exhibited fluctuations comparable to those observed in both the SDH–Boscalid and SDH–Fluxapyroxad complexes, indicating that all three complexes displayed similar dynamic behaviors. This observation suggested that the binding of each ligand to the SDH protein induced a comparable level of structural flexibility, thereby confirming that the ligand–protein interactions were not only stable but also consistent across the board. Such behavior was vital for understanding the long‐term stability of these complexes and their potential performance in a biological context. Furthermore, the RMSF analysis revealed that the binding of compound 6j, Boscalid, and Fluxapyroxad to the active site of the SDH protein resulted in minimal fluctuations among key residues at the binding interface. This stability indicated that each ligand contributed to a robust interaction with the SDH protein, preserving the integrity of the protein structure throughout the simulation. Importantly, the stabilization effects exerted by these ligands could prove essential in enhancing their binding affinity to the SDH protein, which may subsequently increase the efficacy of these compounds in modulating SDH activity. By reducing the fluctuations in the protein structure, the ligands likely facilitated a more effective and durable interaction, thereby contributing to the overall potency of these compounds. This stabilization of the SDH protein was particularly significant, as it had the potential to greatly enhance the biological activity of the compounds, positioning them as promising candidates for the development of future SDHIs.

Figure 7.

(A) RMSDs of all atoms of complexes SDH–Boscalid, SDH–Fluxapyroxad and SDH–6j during 50 ns simulation. (B) RMSFs of residues of complexes SDH–Boscalid, SDH–Fluxapyroxad and SDH–6j during 50 ns simulation. Decomposition of binding energy on the per‐residue basis in complexes SDH–6j (C), SDH–Boscalid (D) and SDH–Fluxapyroxad (E).

The free energy of binding between a protein and its ligand was a crucial metric for evaluating the strength and stability of their interactions. In molecular interactions, a more negative free energy value typically signifies a stronger binding affinity, which was essential for the effective design of inhibitors. ⁴⁴ In this study, we calculated the relative binding energies for the SDH–6j complex, revealing a value of −26.44 kcal mol⁻¹. This value was more negative than SDH–Boscalid (−25.88 kcal mol⁻¹) while was close to SDH–Fluxapyroxad (−32.29 kcal mol⁻¹). These findings indicated that the binding affinity of SDH–6j was stronger than Boscalid and comparable to that of Fluxapyroxad. This high affinity positions compound 6j as a promising candidate for further development in the quest for effective SDHIs. A detailed analysis of the contributions to the binding free energies from specific amino acid residues within the active sites of the SDH–6j (Fig. 7(C)), SDH–Boscalid (Fig. 7(D)), and SDH–Fluxapyroxad (Fig. 7(E)) complexes provided fascinating insights into the underlying mechanisms of binding. Notably, both van der Waals interactions (ΔE _vdw) and the majority of electrostatic interactions (ΔE _ele) contributed negatively to the overall binding energy, highlighting their critical role in stabilizing the presence of small molecules within the enzyme's active pocket. Van der Waals forces facilitated close physical proximity between the ligand and the receptor, while electrostatic interactions enhanced charge‐based attractions, further strengthening the binding affinity. Conversely, the positive value of polar solvation energy (ΔE _psol) suggested that polar solvation adversely affects ligand binding. This indicated the potential presence of water molecules or other polar components in the surrounding environment, which might hinder the proper accommodation of the ligand within the receptor's active site. In contrast, the negative value of non‐polar solvation energy (ΔE _Npsol) implied that interactions with hydrophobic regions of the receptor support ligand binding by promoting a more stable overall structure of the complex. This delicate balance between polar and non‐polar interactions underscored the complexity of ligand binding and emphasizes the need to carefully consider the physicochemical properties of potential inhibitors. Furthermore, the contributions of specific residues to the binding energies were particularly noteworthy. The ΔE _vdw and ΔE _total contributions from the residue TYR91 were significantly higher in the SDH–6j complex compared to both the SDH–Boscalid and SDH–Fluxapyroxad complexes. Meanwhile, the ΔE _ele contributions from the residue TRP173 in the SDH–6j complex was higher than in the SDH–Boscalid complex but much lower than those in the SDH–Fluxapyroxad complex. The distinct differences in the binding processes of compound 6j to SDH provided valuable insights that could inform strategies for delaying or mitigating the development of resistance to SDHIs. Overall, these findings reveal critical information about the binding dynamics and interactions at play, guiding future efforts in the design of more effective SDHIs.

3.7. DFT and ESP calculation

DFT and ESP calculations were conducted to gain a more comprehensive understanding of the antifungal mechanisms and structural characteristics of the target compounds. The frontier molecular orbitals (FMOs), including the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), and the energy gap (ΔE), for compound 6j and the reference fungicide Fluxapyroxad are presented in Fig. 8(A). According to FMO theory, a smaller HOMO–LUMO energy gap generally corresponds to higher chemical reactivity and, in the context of agrochemicals, is often associated with enhanced biological activity. ⁴⁵ In this study, the calculated energy gaps followed the order: compound 6j (ΔE = 4.47 eV) < Fluxapyroxad (ΔE = 5.24 eV), which was consistent with the observed superior fungicidal activity of compound 6j. Analysis of the FMOs revealed distinct electron distribution patterns that provided mechanistic insights. For compound 6j, the HOMO was predominantly localized on the indoline moiety, while the LUMO was primarily distributed across the pyrazole ring, the amide bridge, and the benzyl group linked to the indoline. This arrangement indicated that electron transfer in compound 6j occurred from the indoline group to the pyrazole ring and benzyl substituent, mediated by the amide bridge, which might facilitate interactions with the target SDH enzyme. Similarly, the HOMO of Fluxapyroxad was mainly concentrated on the diphenyl group and amide bond, whereas the LUMO was also centered on these groups with partial extension to the pyrazole ring. This suggested that in Fluxapyroxad, electron transition occurred from the diphenyl moiety to the pyrazole ring via the amide linkage. Collectively, these observations indicated that the electron distribution and transition pathways of compound 6j closely resemble those of Fluxapyroxad, supporting its potential as a highly active SDHI. Complementary ESP analyses were performed to map the regions of electron density and electrostatic potential (Fig. 8(B)). In these maps, red regions represent negative electrostatic potentials, which are prone to attracting electrophiles and often correspond to bioactive sites, whereas blue regions indicate positive electrostatic potentials. ⁴⁶ , ⁴⁷ For both compound 6j and Fluxapyroxad, the regions of strongest negative electrostatic potential were primarily located at the F atom of the CHF₂ group, the oxygen atom of the amide bond, and the nitrogen atom of the pyrazole ring. This distribution highlighted the critical roles of these functional groups in mediating antifungal activity, likely by engaging in key interactions with the active site of SDH. Overall, the combined insights from DFT and ESP calculations provided a detailed picture of the electronic properties and reactive sites of compound 6j. These findings not only rationalized its potent antifungal activity but also underscored its structural and electronic similarities to Fluxapyroxad. As such, compound 6j represented a promising addition to the SDHI family, offering valuable guidance for the design and development of next‐generation fungicides with improved efficacy and selectivity.

Figure 8.

DFT (A) and ESP (B) of compound 6j and Fluxapyroxad.

4. CONCLUSION

In summary, 30 pyrazole‐4‐carboxamide derivatives containing indoline moiety were successfully designed and synthesized, with their antifungal activities against five major plant pathogenic fungi evaluated. The results revealed that most of the target compounds with a benzyl group demonstrated exceptional antifungal potencies. Notably, compound 6j exhibited the most outstanding antifungal activity against R. solani with an impressive EC₅₀ of 0.04 μg mL⁻¹, superior to those of Boscalid (EC₅₀ = 0.40 μg mL⁻¹) and Fluxapyroxad (EC₅₀ = 0.06 μg mL⁻¹). Compound 6i displayed the most potent antifungal activity against S. sclerotiorum and V. mali with EC₅₀ values of 1.79 μg mL⁻¹ and 3.54 μg mL⁻¹, respectively. Further in vivo fungicidal tests demonstrated that compound 6j effectively suppressed the growth of R. solani on rice leaves and compound 6i could suppressed the growth of S. sclerotiorum on oilseed rape leaves and V. mali on apple fruits. These findings suggest that compounds 6j and 6i hold significant potential for the control of R. solani, S. sclerotiorum, and V. mali, presenting promising candidates for crop protection. In addition, detailed mechanistic studies, including SEM analysis, SDH enzyme inhibition assays, molecular docking, MD simulations, DFT and ESP calculations revealed that compound 6j shared a similar antifungal mechanism with Fluxapyroxad. The promising in vitro and in vivo results, along with the detailed mechanistic insights, strongly suggested that compounds 6j and 6i could serve as valuable lead compounds in the development of novel SDHI fungicides for crop protection. Further research, including field trials and optimization of the chemical structures, are warranted to fully realize the potential of compounds 6j and 6i as new, effective tools for protection of rice, oilseed rape and apple fruits.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Method S1. Instruments and chemicals.

Method S2. In vitro antifungal activities.

Method S3. In vivo antifungal activities.

Method S4. In vitro SDH inhibition assay.

Method S5. Molecular docking.

Method S6. Molecular dynamics simulation.

Data S1. The physical data of target compounds.

Data S2. ¹H NMR, ¹³C NMR, and HRMS (ESI) spectra of target compounds.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.