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. 2026 Sep 27;23(9):e71784. doi: 10.1002/cbdv.71784

Design, Synthesis, and Biological Evaluation of Pyrazol‐5‐yl‐Phenylacetamide Derivatives as Potential Fungicidal Agents

Guo‐Tai Lin 1,2,#, Wei Wang 2,3,#, Xi‐Zhe Zhang 2, Li Luo 2, Gong Xu 1,2, Dan Xu 1,2,✉
PMCID: PMC13616471  PMID: 42801763

ABSTRACT

Fungal infestation in crops can greatly reduce both yield and quality. To discover new fungicides, pyrazol‐5‐yl‐phenylacetamide derivatives were synthesized by elongating the carbon chain of the lead compound pyrazol‐5‐yl‐benzamides. These derivatives were assessed for their effectiveness against fungi. Compound C‐13 displayed significant antifungal activity against Sclerotinia sclerotiorum, with an EC50 value of 2.51 mg/L. Additionally, compound C‐16 showed effective antifungal properties against Valsa mali, with an EC50 value of 3.14 mg/L. Particularly noteworthy is the superior antifungal efficacy of compound C‐42 against Gaeumannomyces graminis, with an EC50 value of 1.81 mg/L, significantly outperforming Fluxapyroxad (EC50 = 21.01 mg/L). Molecular docking and SDH enzymatic inhibition assay results provided insights into the potential mechanism of action of compound C‐42. This study supports the development of pyrazol‐5‐yl‐phenylacetamides as promising fungicides, particularly against G. graminis.

Keywords: antifungal activity, Gaeumannomyces graminis, pyrazolamide, structure‐activity relationships

The extension of pyrazol‐5‐yl‐benzamides leads to the development of novel phenylacetamides with exceptional efficacy, particularly against Gaeumannomyces graminis. Compound C‐42 demonstrates an EC50 of 1.81 mg/L, surpassing the commercial SDHI fluxapyroxad by over tenfold. Enzymatic assays confirm SDH as the specific target. This study highlights pyrazol‐5‐yl‐phenylacetamides as promising fungicides for controlling take‐all disease.

graphic file with name CBDV-23-e71784-g003.webp

1. Introduction

Crop losses caused by phytopathogenic fungi remain a serious challenge in global agriculture [1, 2, 3]. Take‐all, caused by the soilborne fungus Gaeumannomyces graminis, is among the most destructive wheat root diseases worldwide, resulting in major losses in yield and quality [4, 5]. Although it continues to be a focus of intensive research, take‐all remains the most important root disease of wheat globally [6]. In recent years, succinate dehydrogenase inhibitors (SDHIs) have attracted considerable interest due to their potent and broad‑spectrum antifungal properties [7, 8, 9, 10, 11, 12, 13]. Currently, 25 SDHI products are commercially available. However, the overuse and inherent target specificity of the single ubiquinone‑binding site [14, 15, 16, 17, 18, 19] have facilitated the emergence of resistant fungal populations [20, 21, 22, 23, 24].

In recent years, significant research efforts have focused on the development of innovative SDHIs [25]. Various effective strategies, such as scaffold hopping [26], bioisosterism [27], and virtual screening [16], have been extensively utilized, leading to the discovery of novel and highly potent SDHI candidates. A common approach for lead optimization involves extending the carbon chain to adjust its flexibility and create a target molecule with a non‐planar shape compared to the lead compound (Figure 1a,b) [15, 28, 29, 30]. This modification has the potential to alter the binding mode with the target sites, offering benefits in addressing the escalating issue of resistance and broadening the spectrum of fungal control.

FIGURE 1.

FIGURE 1

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Design strategy of target compounds through carbon chain extension.

Our previous studies have shown that pyrazol‐5‐yl‐benzamides represent a promising class of lead compounds for SDHI development [31, 32, 33, 34]. In this work, a series of novel pyrazol‐5‐yl‐phenylacetamide derivatives were designed and synthesized utilizing the carbon chain extension strategy (Figure 1c). The in vitro antifungal activity of the synthesized compounds was evaluated against six common phytopathogenic fungi/oomycetes, followed by structure–activity relationship (SAR) analysis. To gain insight into the mode of action, scanning electron microscopy (SEM), molecular docking, and SDH enzyme inhibition assays were additionally performed.

2. Results and Discussion

2.1. Synthesis

Bioactivity‐guided synthesis was utilized to identify fungicidal candidates by investigating the structural features of target compounds. This study was divided into four parts. First, compounds C‐1−C‐16 were synthesized to assess the structure‐activity relationship of the substituents on the phenylacetic acid moiety. These compounds featured a single substitution on the phenylacetic acid moiety and a trifluoromethyl‐substituted pyrazole ring at the third position. Second, compounds C‐17−C‐32 were synthesized to investigate the influence of difluoromethyl substituents at the third position of the pyrazole on the fungicidal activities. The third part involved investigating the effect of multiple substituents or replacement of the phenylacetic acid moiety on the fungicidal activity, leading to the synthesis of compounds C‐33−C‐38. Last, the fourth part focused on examining the introduction of a fluorine or chlorine atom at the fourth position of the pyrazole ring and determining the combined effect of the substituents at third and fourth positions of the phenylacetic acid moiety on the fungicidal activity, resulting in the synthesis of compounds C‐39−C‐50.

The synthetic routes for the target compounds C‐1−C‐50 were presented in Scheme 1. The chemical structures of all the synthesized compounds were confirmed by 1H NMR, 13C NMR, and HRMS analyses, and the related characterization data can be found in Figures S1–S150.

SCHEME 1.

SCHEME 1

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General synthesis of compounds C‐1–C‐50.

2.2. In Vitro Fungicidal Activities

In order to identify target compounds with effective fungicidal activity, six different substituents (hydrogen, methyl, methoxy, chlorine, fluorine and trifluoromethyl) at three different positions (ortho, meta, and para) on the phenylacetic acid moiety were considered in the design of the target compounds. Consequently, the target compounds C‐1−C‐16 were synthesized, and a preliminary screening was conducted for their fungicidal activity at a concentration of 50 mg/L. The results presented in Table 1 demonstrate significant antifungal activity of several compounds against Sclerotinia sclerotiorum, Valsa mali, and G. graminis. Compounds C‐13 (R2 = 4‐Cl, 92.3%), C‐15 (R2 = 3‐CF3, 80.8%), and C‐16 (R2 = 4‐CF3, 91.3%) exhibited inhibitory rates exceeding 80% against S. sclerotiorum. Similarly, compounds C‐9 (R2 = 3‐F, 80.6%), C‐13 (R2 = 4‐Cl, 80.6%), and C‐16 (R2 = 4‐CF3, 83.7%) displayed inhibitory rates surpassing 80% against V. mali. Additionally, compounds C‐12 (R2 = 3‐Cl, 96.6%), C‐13 (R2 = 4‐Cl, 98.7%), C‐15 (R2 = 3‐CF3, 88.6%), and C‐16 (R2 = 4‐CF3, 86.2%) demonstrated inhibitory rates of more than 85% against G. graminis. Notably, compounds C‐13 (R2 = 4‐Cl) and C‐16 (R2 = 4‐CF3) showed broad‐spectrum antifungal activity. A comparison of the inhibition rates of C‐11 (R2 = 2‐Cl, 69.7%, 62.3%, 44.6%, 44.5%), C‐12 (R2 = 3‐Cl, 79.6%, 73.4%, 96.6%, 73.8%), C‐13 (R2 = 4‐Cl, 92.3%, 80.6%, 98.7%, 71.4%), C‐14 (R2 = 2‐CF3, 71.5%, 64.3%, 33.8%, 42.4%), C‐15 (R2 = 3‐CF3, 80.8%, 76.0%, 88.6%, 55.7%), and C‐16 (R2 = 4‐CF3, 91.3%, 83.7%, 86.2%, 59.5%) against S. sclerotiorum, V. mali, G. graminis, and Rhizoctonia solani, respectively, highlights the favorable activity of target compounds bearing a para‐substituted phenylacetic acid moiety, particularly with chlorine or trifluoromethyl substituents on the phenyl ring.

TABLE 1.

In vitro fungicidal activities (inhibition rate) of compounds C‐1−C‐50 at 50 mg/L a .

Compd. R1 R2 R3 S.s. b P.c. c V. m. d B.c. e G.g. f R.s. g
C‐1 CF3 H H 50.9 ± 1.2 25.5 ± 2.0 37.8 ± 1.2 <5 75.2 ± 1.1 36.2 ± 1.6
C‐2 CF3 2‐CH3 H 58.8 ± 0.3 19.3 ± 1.9 50.1 ± 0.6 12.3 ± 2.0 36.8 ± 2.0 31.5 ± 1.2
C‐3 CF3 3‐CH3 H 41.2 ± 1.5 21.2 ± 2.2 32.7 ± 1.5 <5 48.7 ± 1.6 75.7 ± 0.6
C‐4 CF3 4‐CH3 H 61.2 ± 0.9 13.5 ± 1.8 33.7 ± 1.6 35.1 ± 1.3 49.8 ± 1.2 37.6 ± 2.2
C‐5 CF3 2‐OCH3 H 33.3 ± 2.1 31.5 ± 2.3 54.1 ± 1.2 <5 46.3 ± 1.3 29.5 ± 3.1
C‐6 CF3 3‐OCH3 H 55.2 ± 1.6 <5 31.7 ± 1.7 18.3 ± 2.0 53.7 ± 0.5 34.2 ± 0.6
C‐7 CF3 4‐OCH3 H 38.1 ± 1.2 13.7 ± 3.5 37.4 ± 2.0 20.5 ± 1.0 58.1 ± 0.6 31.6 ± 1.1
C‐8 CF3 2‐F H 33.9 ± 2.3 <5 36.8 ± 1.6 <5 50.4 ± 1.1 26.5 ± 2.9
C‐9 CF3 3‐F H 56.3 ± 0.5 14.5 ± 3.3 80.6 ± 0.7 5.3 ± 2.3 33.8 ± 1.6 33.1 ± 2.0
C‐10 CF3 4‐F H 46.1 ± 1.9 <5 39.9 ± 1.2 21.5 ± 3.6 32.2 ± 2.0 26.0 ± 1.3
C‐11 CF3 2‐Cl H 69.7 ± 1.2 11.6 ± 2.1 62.3 ± 2.0 31.8 ± 1.6 44.6 ± 1.6 44.5 ± 2.2
C‐12 CF3 3‐Cl H 79.6 ± 1.0 44.3 ± 1.5 73.4 ± 1.3 19.4 ± 2.0 96.6 ± 0.1 73.8 ± 1.1
C‐13 CF3 4‐Cl H 92.3 ± 0.6 48.1 ± 0.6 80.6 ± 1.2 30.2 ± 2.2 98.7 ± 0.2 71.4 ± 0.4
C‐14 CF3 2‐CF3 H 71.5 ± 0.8 12.6 ± 3.3 64.3 ± 0.6 14.0 ± 1.3 33.8 ± 1.2 42.4 ± 1.3
C‐15 CF3 3‐CF3 H 80.8 ± 1.1 41.2 ± 1.8 76.0 ± 1.2 42.5 ± 2.0 88.6 ± 0.3 55.7 ± 1.2
C‐16 CF3 4‐CF3 H 91.3 ± 0.2 39.5 ± 1.2 83.7 ± 1.3 30.2 ± 2.2 86.2 ± 0.6 59.5 ± 1.6
C‐17 CHF2 H H 35.2 ± 2.3 <5 33.7 ± 0.8 <5 48.7 ± 1.1 28.6 ± 2.8
C‐18 CHF2 2‐CH3 H 39.5 ± 2.5 14.5 ± 3.2 24.6 ± 1.1 15.6 ± 2.3 34.7 ± 1.5 22.2 ± 3.1
C‐19 CHF2 3‐CH3 H 42.3 ± 1.6 11.3 ± 2.0 42.5 ± 2.0 <5 32.5 ± 2.2 35.1 ± 1.2
C‐20 CHF2 4‐CH3 H 56.8 ± 0.6 <5 74.5 ± 1.5 <5 66.9 ± 1.3 23.5 ± 1.6
C‐21 CHF2 2‐OCH3 H 44.9 ± 0.7 <5 41.9 ± 1.6 <5 41.3 ± 2.1 12.6 ± 2.1
C‐22 CHF2 3‐OCH3 H 69.7 ± 1.1 12.6 ± 3.3 43.9 ± 1.8 14.5 ± 1.6 17.3 ± 1.9 24.2 ± 3.1
C‐23 CHF2 4‐OCH3 H 35.8 ± 2.1 <5 43.9 ± 1.2 15.6 ± 2.2 58.6 ± 0.8 28.6 ± 3.5
C‐24 CHF2 2‐F H <5 <5 62.3 ± 1.1 <5 34.7 ± 1.3 19.5 ± 2.6
C‐25 CHF2 3‐F H 26.7 ± 2.6 <5 27.6 ± 2.0 <5 29.7 ± 2.1 21.2 ± 3.1
C‐26 CHF2 4‐F H <5 <5 40.9 ± 1.6 <5 54.5 ± 0.6 11.4 ± 3.6
C‐27 CHF2 2‐Cl H 39.4 ± 1.5 14.5 ± 1.5 21.5 ± 2.0 12.9 ± 1.1 41.3 ± 1.6 26.6 ± 2.6
C‐28 CHF2 3‐Cl H 71.3 ± 1.2 12.6 ± 1.3 46.2 ± 0.7 31.5 ± 1.3 28.9 ± 2.2 35.2 ± 2.3
C‐29 CHF2 4‐Cl H 64.9 ± 1.0 10.2 ± 1.5 46.0 ± 1.5 23.2 ± 0.6 28.0 ± 1.3 36.6 ± 1.6
C‐30 CHF2 2‐CF3 H 32.1 ± 2.2 13.5 ± 1.2 86.8 ± 0.8 <5 74.4 ± 1.8 28.6 ± 2.2
C‐31 CHF2 3‐CF3 H 12.1 ± 3.5 33.3 ± 2.0 32.3 ± 2.0 26.1 ± 2.3 30.8 ± 2.1 45.2 ± 2.1
C‐32 CHF2 4‐CF3 H 78.2 ± 1.0 25.1 ± 1.8 46.0 ± 1.6 38.9 ± 1.8 46.2 ± 1.3 41.2 ± 1.6
C‐33 CF3 2,4‐diCl H 32.8 ± 0.9 25.7 ± 2.3 63.1 ± 1.6 15.3 ± 3.1 30.8 ± 1.2 71.6 ± 2.0
C‐34 CF3 3,4‐diCl H 47.0 ± 1.2 35.2 ± 1.6 61.1 ± 2.0 19.8 ± 2.6 40.8 ± 0.9 48.5 ± 1.1
C‐35 CF3 3,5‐diCl H 84.3 ± 0.6 56.2 ± 0.8 83.3 ± 1.6 57.7 ± 1.3 45.8 ± 1.6 48.7 ± 0.8
C‐36 CF3 2,4‐diCF3 H 14.1 ± 3.2 23.8 ± 2.0 33.3 ± 2.3 31.5 ± 2.0 33.3 ± 2.0 58.1 ± 1.2
C‐37 CF3 3,5‐diCF3 H 11.1 ± 3.6 33.3 ± 1.5 17.2 ± 2.1 25.2 ± 1.2 28.3 ± 1.9 34.7 ± 1.6
C‐38 CF3 — H <5 21.9 ± 2.0 27.8 ± 1.9 17.1 ± 1.6 28.1 ± 2.1 36.5 ± 2.0
C‐39 CF3 3‐Cl Cl 65.7 ± 0.8 47.6 ± 1.1 77.8 ± 0.8 33.3 ± 1.3 55.8 ± 1.3 77.0 ± 1.3
C‐40 CF3 4‐Cl Cl 56.1 ± 1.2 69.5 ± 0.6 78.3 ± 0.6 57.7 ± 1.1 98.3 ± 0.2 55.4 ± 1.3
C‐41 CF3 3‐Cl F 32.8 ± 2.0 39.1 ± 1.2 63.6 ± 1.2 23.4 ± 0.6 41.7 ± 1.2 85.1 ± 0.9
C‐42 CF3 4‐Cl F 60.6 ± 1.5 53.3 ± 0.7 86.9 ± 0.6 35.1 ± 1.3 100 58.1 ± 1.2
C‐43 CF3 3‐CF3 Cl 28.8 ± 2.2 32.4 ± 1.6 43.4 ± 2.3 25.2 ± 1.6 34.2 ± 1.1 74.8 ± 1.1
C‐44 CF3 4‐CF3 Cl 27.8 ± 1.8 35.2 ± 1.5 61.1 ± 0.7 26.1 ± 2.0 53.3 ± 0.9 48.7 ± 0.6
C‐45 CF3 3‐CF3 F 26.9 ± 2.6 22.0 ± 2.0 46.0 ± 1.6 30.8 ± 1.6 33.3 ± 2.1 66.2 ± 0.9
C‐46 CF3 4‐CF3 F 33.8 ± 1.2 29.5 ± 1.6 29.3 ± 2.0 20.7 ± 2.6 30.8 ± 1.3 50.5 ± 1.2
C‐47 CF3 3‐CH3 Cl 51.0 ± 1.6 55.2 ± 0.7 80.8 ± 0.6 45.1 ± 1.3 62.5 ± 0.9 77.0 ± 1.3
C‐48 CF3 4‐CH3 Cl 31.3 ± 2.5 43.8 ± 1.1 71.7 ± 0.7 39.6 ± 2.0 55.8 ± 1.1 56.3 ± 1.2
C‐49 CF3 3‐CH3 F 33.3 ± 2.3 56.2 ± 0.6 62.1 ± 1.6 23.4 ± 1.6 53.3 ± 1.2 92.8 ± 0.2
C‐50 CF3 4‐CH3 F 44.9 ± 1.1 38.0 ± 2.1 32.9 ± 2.0 22.2 ± 2.6 36.7 ± 0.6 45.7 ± 1.2
Fluxapyroxad 100 28.9 ± 1.6 59.2 ± 0.9 25.3 ± 2.1 65.0 ± 1.1 89.2 ± 1.6
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a

Data are given as the mean of triplicate experiments.

b

S. sclerotiorum.

c

P. capsici.

d

V. mali.

e

B. cinerea.

f

G.graminis.

g

R. solani.

To investigate the impact of trifluoromethyl and difluoromethyl substituents at the third position of the pyrazole ring on the biological activity of the target compounds, a series of compounds C‐17−C‐32 bearing difluoromethyl substituents were synthesized and compared with compounds C‐1−C‐16 containing trifluoromethyl substituents. The results revealed that trifluoromethyl substituents on the pyrazole ring could significantly enhance the fungicidal activities of the target compounds. For instance, compound C‐13 (R1  = CF3, R2 = 4‐Cl) exhibited superior activity compared to C‐29 (R1  = CHF2, R2 = 4‐Cl), while compound C‐16 (R1  = CF3, R2 = 4‐CF3) demonstrated higher efficacy than C‐32 (R1  = CHF2, R2 = 4‐CF3) against S. sclerotiorum, V. mali, G. graminis, Phytophthora capsici, and R. solani.

The screening of the target compounds C‐1−C‐32 initially identified compounds C‐13 (R2 = 4‐Cl) and C‐16 (R2 = 4‐CF3) as displaying notable fungicidal activity. Subsequently, to evaluate the impact of polysubstituted phenylacetic acid moiety on fungicidal activity, compounds C‐33 (R2 = 2, 4‐diCl), C‐34 (R2 = 3, 4‐diCl), C‐35 (R2 = 3, 5‐diCl), C‐36 (R2 = 2, 4‐diCF3), C‐37 (R2 = 3, 5‐diCF3), and C‐38 (Ar‐R2 = naphthalen‐2‐yl) were synthesized. The results indicated that compounds with di‐substituted benzoic acid moiety exhibited significantly lower fungicidal activity compared to those with mono‐substituted benzoic acid moiety. For instance, compounds C‐33 (R2 = 2, 4‐diCl) and C‐34 (R2 = 3, 4‐diCl) showed reduced activity relative to C‐13 (R2 = 4‐Cl), while compounds C‐36 (R2 = 2, 4‐diCF3) and C‐37 (R2 = 3, 5‐diCF3) demonstrated decreased activity compared to C‐16 (R2 = 4‐CF3). Additionally, the replacement of phenylacetic acid moiety in compound C‐1 with naphthalen‐2‐ylacetic acid moiety resulted in compound C‐38 (Ar‐R2 = naphthalen‐2‐yl) exhibiting a significant decrease in antifungal activity.

The target compounds were further modified by the introduction of fluorine or chlorine atom at the 4‐position of the pyrazole. A series of compounds C‐39−C‐50 containing a 4‐substituted benzoic acid moiety (including chlorine, trifluoromethyl, and methyl substitutions) were designed and synthesized based on the previous experience. The results indicated significant variations in inhibitory effects on different fungi. For S. sclerotiorum and V. mali, the antifungal activity generally decreased after introducing a fluorine or chlorine atom at the 4‐position of the pyrazole. For example, comparing compound C‐13 (R1  = CF3, R2 = 4‐Cl, R3 = H) with compounds C‐40 (R1  = CF3, R2 = 4‐Cl, R3 = Cl) and C‐42 (R1  = CF3, R2 = 4‐Cl, R3 = F), the antifungal activity decreased. Similarly, when comparing compound C‐16 (R1  = CF3, R2 = 4‐CF3, R3 = H) with compounds C‐44 (R1  = CF3, R2 = 4‐CF3, R3 = Cl) and C‐46 (R1  = CF3, R2 = 4‐CF3, R3 = F), the antifungal activity also decreased. Conversely, for G. graminis and R. solani, the antifungal activity in some cases increased after introducing a fluorine or chlorine atom at the 4‐position of the pyrazole. For instance, compound C‐16 exhibited an inhibition rate of 86.2% against G. graminis, which increased to 100% and 98.3% after introducing a fluorine (compound C‐42) or chlorine (compound C‐40) atom, respectively. Similarly, compound C‐3 showed an inhibition rate of 75.7% against R. solani, which increased to 92.8% after introducing a fluorine atom (compound C‐49). However, this series of target compounds demonstrated weaker fungicidal activity against Botrytis cinerea and Phytophthora capsici.

The preliminary screening assay identified a number of compounds with good fungicidal activity. To further investigate their antifungal activity and structure‐activity relationship, the effective compounds were selected for testing their EC50 against S. sclerotiorum, V. mali, G. graminis, and R. solani, as outlined in Table 2. Compound C‐13 demonstrated the most potent antifungal activity against S. sclerotiorum with an EC50 of 2.51 mg/L, albeit lower than that of Fluxapyroxad. Additionally, compound C‐16 (R2 = 4‐CF3, EC50 = 3.39 mg/L) exhibited superior efficacy compared to C‐15 (R2 = 3‐CF3, EC50 = 4.63 mg/L). Particularly noteworthy is that compound C‐16 displayed significant activity against V. mali, with an EC50 value of 3.14 mg/L, outperforming Fluxapyroxad (EC50 = 12.45 mg/L). Compounds C‐41 (R2 = 3‐Cl, R3 = F) and C‐49 (R2 = 3‐CH3, R3 = F) showed EC50 values of 4.54 and 2.23 mg/L against R. solani, respectively. Notably, the carbon chain extension strategy led to compound C‑42, which displayed substantially improved activity against G. graminis. It achieved a 100% inhibition rate, far exceeding the 66.2% of the lead compound 9Ig (Table S1). Moreover, compound C‐42 (R2 = 4‐Cl, R3 = F) demonstrated exceptional antifungal activity against G. graminis, with an EC50 of 1.81 mg/L, surpassing that of Fluxapyroxad (EC50 = 21.01 mg/L). These results collectively validate the rationality of the carbon chain extension design.

TABLE 2.

In vitro EC50 values (mg/L) of the selected compounds.

Fungi Compd. EC50 95% CI a Regression equation R 2
S. sclerotiorum C‐13 2.51 1.26–3.86 y = −0.262 + 0.657x 0.941
C‐15 4.63 2.94–6.54 y = −0.467 + 0.702x 0.931
C‐16 3.39 2.30–4.54 y = −0.488 + 0.920x 0.925
C‐35 5.20 4.05–6.48 y = −0.820 + 1.144x 0.972
Fluxapyroxad 0.55 0.20–0.98 y = 0.253 + 0.974x 0.930
V. mali C‐9 3.84 2.21–5.63 y = −0.381 + 0.652x 0.940
C‐13 5.52 3.96–7.30 y = −0.639 + 0.861x 0.962
C‐16 3.14 1.88–4.50 y = −0.226 + 0.397x 0.902
C‐30 4.98 3.52–6.63 y = −0.595 + 0.853x 0.913
C‐35 6.19 4.30–8.49 y = −0.597 + 0.754x 0.928
C‐42 6.31 4.81–8.07 y = −0.790 + 0.987x 0.945
C‐47 4.05 2.67–5.55 y = −0.486 + 0.800x 0.989
Fluxapyroxad 12.45 9.49–18.66 y = −1.243 + 1.135x 0.993
R. solani C‐41 4.54 3.16–6.07 y = −0.561 + 0.854x 0.924
C‐49 2.23 1.37–3.14 y = −0.318 + 0.913x 0.948
Fluxapyroxad 0.62 0.35–0.88 y = 0.224 + 1.070x 0.981
G. graminis C‐12 13.88 7.55–65.56 y = −0.487 + 0.426x 0.958
C‐13 2.08 0.40–3.85 y = −0.147 + 0.462x 0.930
C‐15 6.20 4.04–9.50 y = −0.519 + 0.655x 0.976
C‐16 4.57 2.99–6.44 y = −0.497 + 0.753x 0.915
C‐40 2.71 1.19–4.25 y = −0.264 + 0.608x 0.916
C‐42 1.81 0.64–2.99 y = −0.162 + 0.630x 0.901
Fluxapyroxad 21.01 17.7–25.8 y = −2.411 + 1.822x 0.970
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a

Confidence interval.

2.3. Scanning Electron Microscopy (SEM) Analysis

SEM was used to examine the morphological changes in the hyphae of S. sclerotiorum and V. mali (Figure 2). In the untreated control groups, the hyphae of S. sclerotiorum (A, C) and V. mali (E, G) appeared regular, smooth, and consistent in thickness and fullness, with a high density. When treated with compounds C‐13 and C‐16 at a concentration of 5 mg/L, respectively, a significant decrease in hyphal density was observed for both species. Specifically, the hyphae of S. sclerotiorum (B, D) showed surface shrinkage, dry cracks, and extreme unevenness in thickness, while the hyphae of V. mali (F, H) displayed wrinkles, severe distortion in individual hyphae, and significant variations in thickness. These results demonstrate that compounds C‐13 and C‐16 have detrimental and inhibitory effects on the morphology and growth of S. sclerotiorum and V. mali hyphae, respectively.

FIGURE 2.

FIGURE 2

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Scanning electron micrographs of the hyphae from the colonies of S. sclerotiorum and V. mali.

2.4. Molecular Docking

Docking simulations were conducted for compounds C‑13, C‑16, C‑42, and Fluxapyroxad using the crystal structure of SDH (PDB: 2FBW) to probe their binding modes and possible mechanism of action. The results, depicted in Figure 3, revealed that upon carbon chain extension, both the amino and carboxylic acid moieties of the compounds interacted with the active pocket of SDH. Specifically, the phenylacetic acid segments of compounds C‐13, C‐16, and C‐42 occupied the left pocket of the active site, while the pyrazole ring section entered the right pocket. In contrast, Fluxapyroxad exclusively featured the pyrazole ring interacting with the right pocket of the active site. Additionally, the binding energies of compounds C‐13, C‐16, C‐42, and Fluxapyroxad were determined to be −41.27, −41.06, −39.47, and −41.09 kcal/mol, respectively, showing relatively similar values.

FIGURE 3.

FIGURE 3

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Binding models of C‐13 (A), C‐16 (B), C‐42 (C), and Fluxapyroxad (D) in the active site of SDH (PDB entry: 2FBW).

As shown in Figure 3 (A2–D2, A3–D3), the tested compounds engage with multiple amino acid residues within the binding pocket, contributing to the stabilization of their binding conformations. All compounds (C‐13, C‐16, C‐42, and Fluxapyroxad) engage in p‐π conjugation with Arg 43, enhancing their stability within the binding site. Notably, compounds C‐13 and C‐16 form hydrogen bonds with Trp 173 through the oxygen atoms of the amide bond, while compound C‐42 and Fluxapyroxad establish hydrogen bonds with Trp 173 and Tyr 58, respectively, via the nitrogen atom of the amide group. Furthermore, compound C‐42 forms an additional hydrogen bond between its fluorine atom at the fourth position of the pyrazole ring and Ser 39. Although compounds C‐13, C‐16, C‐42, and Fluxapyroxad exhibit similarities in their binding modes with SDH, differences in binding modes and energies are evident among them.

2.5. SDH Enzyme Assay In Vitro

To gain further insight into its antifungal mechanism, compound C‐42, which showed the best in vitro activity against G. Graminis (EC50 = 1.81 mg/L), was evaluated for SDH inhibition. As summarized in Table 3, C‐42 inhibited SDH with an IC50 of 18.33 mg/L, indicating moderate inhibitory activity compared to Fluxapyroxad (IC50 = 7.28 mg/L).

TABLE 3.

SDH inhibition activity (IC50).

Compd. IC50 (mg/L) 95% CI a Regression equation R 2
C‐42 18.33 10.89−41.88 y = −0.767 + 0.608x 0.973
Fluxapyroxad 7.28 5.18−11.38 y = −0.622 + 0.721x 0.994
Open in a new tab
a

Confidence interval.

3. Conclusions

In summary, a series of novel pyrazol‐5‐yl‐phenylacetamide derivatives were synthesized and evaluated for their efficacy against various fungi. Several compounds, including C‐13, C‐16, and C‐42, demonstrated significant antifungal activity against S. sclerotiorum, V. mali, and G. graminis, respectively. Notably, compound C‐42 displayed exceptional antifungal properties against G. graminis, with an EC50 value of 1.81 mg/L, surpassing Fluxapyroxad (EC50 = 21.01 mg/L). Molecular docking and SDH enzymatic inhibition assays suggest that these compounds could target SDH. This study provides valuable insights for the potential development of pyrazol‐5‐yl‐phenylacetamides as innovative SDH inhibitors.

3.1. Experimental Section

3.1.1. Materials and Instruments

All reagent‐grade chemicals were procured from commercial sources such as Energy and Adamas. 1H, and 13C NMR spectra were recorded on a Bruker AV‐400 Hz spectrometer, with CDCl3 or DMSO‐d 6 as the solvent and tetramethylsilane as the internal standard. Chemical shifts were reported in ppm (δ). ESI–MS spectra were obtained using a Mariner System 5304 mass spectrometer. Melting points (mp) were measured using a WRS‐1B melting point apparatus (Jingsong, Shanghai, China) without correction. A scanning electron microscope (Hitachi, S‐3400 N, Tokyo, Japan) was employed to observe the microscopic morphology of fungal hyphae. Conductivity was measured by a CON510 Eutech/Oakton conductometer (OAKTON Instruments, Waltham).

3.1.2. Chemistry

The synthetic routes of the target compounds C‐1–C‐50 are shown in Scheme 1. Intermediates 3 and 4 were synthesized from acetonitrile and a substituted ethyl acetate [32], while intermediates 5 and 6 were prepared according to the literature method [33].

3.1.2.1. General Procedure for the Synthesis of Target Compound C‐1–C‐50

Following the methodology described in reference [35], a reaction was performed by dissolving a substituted 2‐phenylacetic acid (1.1 mmol) and intermediate 4I, 4II, 5, or 6 (1 mmol) in 3 mL of dichloromethane (DCM). Subsequently, N‐methylimidazole (3.5 mmol) and N,N,N',N'‐tetramethylchloroformamidinium‐hexafluorophosphate (TCFH, 1.2 mmol) were added to the solution. The reaction mixture was then stirred at room temperature for 4−20 h, and the resulting solution was concentrated to yield the crude product. This crude product was then purified by silica gel chromatography (PE/EtOAc = 5:1) to afford the corresponding target compounds C‐1–C‐50.

3.2. Antifungal Bioassay In Vitro

The fungicidal activities of all synthesized compounds were assessed in vitro against B. cinerea, V. mali, R. solani, S. sclerotiorum, P. capsici, and G. graminis at a concentration of 50 mg/L using the mycelia growth inhibition method [32, 36, 37] for preliminary screening. The detailed experimental method is available in the Supporting Information.

3.3. SEM Analysis

The SEM assay was carried out following the previously described method [32, 36].

3.4. Molecular Docking

Molecular docking was accomplished by employing a method that has been reported [32, 34].

3.5. In Vitro SDH Enzyme Assay

The SDH enzyme activities of C‐42 and Fluxapyroxad were determined using the Mlbio succinate dehydrogenase assay kit (ml076573) and evaluated according to a previously established protocol [32].

Author Contributions

Dan Xu conceived and designed the study, provided overall supervision, and administered the project. Guo‑Tai Lin and Wei Wang contributed to the methodology together with Xi‑Ze Zhang and Li Luo, and also collaborated on data curation and validation. Guo‑Tai Lin and Wei Wang performed the formal analysis, investigation, and software implementation, and were primarily responsible for the visualization of the data and the writing of the original draft. The manuscript was critically reviewed and edited by Xi‑Ze Zhang, Li Luo, Gong Xu, and Dan Xu. Resources were provided by Gong Xu and Dan Xu, while funding acquisition was secured by Wei Wang, Li Luo, and Dan Xu. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71784‐sup‐0001‐SuppMat.docx

CBDV-23-e71784-s001.docx (5.1MB, docx)

Acknowledgments

This research was financially supported by the Natural Science Foundation of Ningxia (2024AAC03138), the National Natural Science Foundation of China (32202355), the Key Laboratory of Integrated Pest Management on the Loess Plateau of Ministry of Agriculture and Rural Affairs (KFJJ202402), and the Undergraduate Training Program for Innovation and Entrepreneurship (XN2025002021). We thank Prof. Zhi‐Qin Ji (Northwest A&F University) for his assistance with computational molecular docking. We also acknowledge Ms. Xiao‐Hua He (Northwest A&F University) for her scanning electron microscopy (SEM) analysis.

Data Availability Statement

The data supporting the findings of this study are available in the Supporting Information of this article, including: 1H NMR, 1 3C NMR, 1 9F NMR, and HRMS (ESI) spectra of the target compounds (Figures S1–S150); physical data of the target compounds; in vitro fungicidal activity assay methods; and the activity data of the lead compound 9Ig against plant pathogens (Table S1).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File 1: cbdv71784‐sup‐0001‐SuppMat.docx

CBDV-23-e71784-s001.docx (5.1MB, docx)

Data Availability Statement

The data supporting the findings of this study are available in the Supporting Information of this article, including: 1H NMR, 1 3C NMR, 1 9F NMR, and HRMS (ESI) spectra of the target compounds (Figures S1–S150); physical data of the target compounds; in vitro fungicidal activity assay methods; and the activity data of the lead compound 9Ig against plant pathogens (Table S1).

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