Scaffold Hopping from Dehydrozingerone: Design, Synthesis, and Antifungal Activity of Phenoxyltrifluoromethylpyridines

Abstract

In response to the urgent need for innovative fungicides to ensure food security and safety, a series of twenty-three novel trifluoromethylpyridine compounds were designed and synthesized using a scaffold hopping strategy derived from dehydrozingerone. This approach involved converting the α, β-unsaturated ketone moiety into a pyridine ring. Bioassay results indicated that the majority of these compounds exhibited promising in vitro antifungal activity, particularly against Rhizoctonia solani and Colletotrichum musae. Notably, compound 17 showed the highest efficacy and broad-spectrum activity, with median effective concentrations (EC₅₀) ranging from 2.88 to 9.09 μg/mL. Phenoxytrifluoromethylpyridine derivatives, including compound 17, exhibited superior antifungal activity compared to benzyloxytrifluoromethylpyridine derivatives. In vivo tests revealed that both compounds 17 and 23 exhibited moderate control effects against C. musae. The degradation half-lives of compounds 17 and 23 in bananas were determined to be 176.9 h and 94.8 h, respectively, indicating the stability of their structures in the environment. Molecular docking studies indicated that compound 23 interacts with succinate dehydrogenase, offering valuable insights for the structural optimization of compound 23.

1. Introduction

Fungicides represent a critical component in the management of plant diseases, which pose a significant challenge to the sustainable development of agriculture [1,2]. However, the emergence and spread of new crop pathogens [3], the development of fungicide resistance [4], and the increasing public demand for food safety and environmental protection have collectively rendered the development of new fungicides an urgent priority [2,5]. Natural products have consistently served as a vital resource for the discovery of new pesticides, and the structural modification of natural compounds is a widely adopted strategy in the pursuit of environmentally friendly pesticides [6,7].

Dehydrozingerone, derived from the ginger plant Aframomum giganteum [8], has garnered significant interest for its diverse pharmacological activities, such as its antioxidant, antimutagenic, anticancer, anti-inflammatory, antidepressant, antimalarial, antiplatelet, Alzheimer’s-disease-preventive, antimicrobial, and β-adrenoceptor antagonist properties [9,10]. In contrast, the application of dehydrozingerone in agriculture is less documented. Notably, it exhibits moderate insect growth-regulatory effects and antifeeding activity against Spodoptera litura, as well as potent antifungal action against Rhizoctonia solani [11]. Moreover, gingerone derivatives have been shown to attract Bactrocera jarvisi, indicating their potential as male chemical lures for integrated pest management [12]. These derivatives also suppress the growth of Aspergillus flavus and Fusarium graminearum and hinder the biosynthesis of their mycotoxins, underscoring their potential as natural food preservatives [13]. In our recent endeavors to develop novel pesticides derived from natural products, we have successfully designed and synthesized a series of derivatives based on dehydrozingerone as the primary scaffold [14,15] and studied their antifungal structure-activity relationships. Notably, the incorporation of electron-withdrawing groups, such as chlorine, on the benzene ring (Part A, Figure 1) significantly contributes to the antifungal properties [14]. Furthermore, the presence of double bonds and ketone carbonyls (Part B, Figure 1) is imperative for the preservation of antifungal activity [11]. Additionally, the introduction of a trifluoromethyl substituent (Part C, Figure 1) is found to be advantageous in augmenting the antifungal potential of these derivatives [14]. We discovered that compound A (Figure 1) exhibits remarkable in vitro antifungal efficacy [14]. However, similar to numerous dehydrozingerone derivatives [15], compound A is unstable and exhibits poor in vivo antifungal activity [12]. To address this, we converted the carbonyl group in compound A to a hydroxyl group, forming compound B (Figure 1), which significantly improved its in vivo activity [15].

Introducing heterocycles into molecules is a crucial method for optimizing pesticide molecules, and trifluoromethylpyridine is a significant heterocycle used extensively in pesticide chemistry, including in the development of herbicides (pyroxsulam), insecticides (pyrionafen), and fungicides (fluopimomide) (Figure 2) [16,17,18]. Succinate dehydrogenase inhibitors (SDHIs) are an important class of fungicides, with the evolution of three generations to date [19,20]. The development of SDHIs has consistently featured a carboxamide core, with the pyrazole-4-carboxamide moiety emerging as a critical and widespread scaffold, and this structural element has been instrumental in enhancing the antifungal efficacy of the developed compounds (isopyrazam, flubeneteram, inpyrfluxam, Figure 2) against plant pathogens [21,22,23,24,25]. In current research, we are advancing the development of novel pesticides based on dehydrozingerone. Inspired by the development process of azoxystrobin and kresoxim-methyl from strobilurin A (Figure 2) [26], we have employed a scaffold hopping strategy [27,28] to transform the α, β-unsaturated ketones of dehydrozingerone into pyridine rings (Figure 2). To facilitate the synthesis, we have incorporated linking arms into both the benzene and pyridine rings (Figure 2). Following this, we synthesized a series of trifluoromethylpyridine derivatives and evaluated their antifungal potencies. The compounds 1~10 were employed to elucidate the impact of the linker and the position of the trifluoromethyl substituent on the pyridine ring on biological activity. Compounds 11~17 were utilized to examine the effects of various substituents on the benzene ring on antifungal efficacy, and compounds 18~23 were developed for further derivatization. Furthermore, we assessed their stability and probed putative targets of compound 23 using molecular docking.

2. Results and Discussion

2.1. Chemistry

Previous findings [14] indicated that the 2,4-dichlorophenyl substituent plays a pivotal role in conferring antifungal activity. In our current work, we first designed the molecule that only changed the position of the trifluoromethyl group in the pyridine ring, as well as the position and length of the linker. We successfully synthesized compounds 1 to 10 via nucleophilic aromatic substitution (SNAr) reactions (Scheme 1) [29]. The synthesis utilized 2,4-dichlorophenol and 2,4-dichlorobenzyl alcohol as starting materials, which were reacted with the appropriate fluoropyridine derivatives. Considering the substantial structural deviations of our designed compounds from dehydrozingerone, we also introduced variations in the substituents on the benzene rings. Employing 2-fluoro-3-(trifluoromethyl)pyridine in conjunction with seven diverse substituted phenolic precursors, we prepared an additional series of compounds 11~17, through SNAr reactions (Scheme 1). Furthermore, compound 17 served as a precursor for the synthesis of a subsequent series, 18~23, by reacting it with a range of acyl chlorides (Scheme 1). The chemical structures of the twenty-three compounds were confirmed by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectra, and the corresponding spectra are provided in the Supplementary Information (Figures S1–S69).

2.2. In Vitro and In Vivo Antifungal Activity

The in vitro antifungal activity of the target compound was tested against six common plant pathogens (Rhizoctonia solani, Pyricularia oryzae, Colletotrichum musae, Fusarium graminearum, Botrytis cinerea, and Colletotrichum siamense). Evaluation was performed at concentrations of 10 and 50 µg/mL, which were selected to accommodate diverse solubility profiles. The results are presented in

Table 1. Inhibitory rate of target compounds on plant pathogen mycelium growth.

Con. a	Compound	Inhibition Rate ± SD (%)
		R. s b	P. o	C. m	F. g	B. c	C. s
50	1	93.5 ± 0.7	79.1 ± 2.4	86.1 ± 0.7	76.8 ± 1.5	90.7 ± 1.8	55.7 ± 3.5
50	2	67.3 ± 2.4	28.6 ± 3.1	47.1 ± 4.1	10.7 ± 7.7	33.2 ± 3.5	23.8 ± 0.7
50	3	86.2 ± 1.1	33.6 ± 3.7	68.4 ± 7.9	58.4 ± 10.6	53.1 ± 14.2	42.9 ± 2.1
50	4	79.1 ± 0.0	59.7 ± 1.4	81.8 ± 0.6	39.1 ± 1.2	15.6 ± 2.1	46.3 ± 4.1
50	5	86.4 ± 0.6	48.1 ± 2.0	84.9 ± 2.8	44.7 ± 3.4	48.3 ± 6.7	51.4 ± 0.7
50	6	74.0 ± 1.0	25.7 ± 5.5	74.3 ± 2.8	28.1 ± 1.4	39.6 ± 1.4	32.4 ± 2.2
50	7	78.8 ± 0.7	30.3 ± 1.8	72.5 ± 7.0	53.7 ± 6.1	17.6 ± 4.5	37.3 ± 4.2
50	8	76.8 ± 1.5	21.1 ± 3.3	65.4 ± 3.6	35.5 ± 3.4	37.9 ± 10.2	26.4 ± 3.0
50	9	91.6 ± 0.0	73.8 ± 0.7	87.0 ± 0.0	67.9 ± 2.5	95.8 ± 1.2	66.7 ± 0.7
50	10	27.2 ± 1.7	8.4 ± 4.6	13.3 ± 2.3	26.1 ± 3.5	13.25 ± 1.0	6.21 ± 2.1
50	11	80.2 ± 1.7	27.5 ± 5.2	70.7 ± 4.5	37.8 ± 23.8	43.0 ± 1.5	29.4 ± 7.1
50	12	86.5 ± 3.7	84.2 ± 6.1	79.8 ± 6.9	78.6 ± 1.5	87.3 ± 3.5	52.8 ± 3.3
10	13	40.1 ± 1.7	25.3 ± 0.7	6.5 ± 2.4	6.3 ± 1.1	14.0 ± 4.0	−0.5 ± 0.7
10	14	59.0 ± 1.2	33.9 ± 3.5	10.2 ± 0.7	15.1 ± 3.4	5.8 ± 6.6	9.7 ± 1.4
10	15	15.0 ± 0.7	5.6 ± 2.6	13.4 ± 3.5	−2.2 ± 1.7	30.3 ± 0.7	4.2 ± 2.7
10	16	60.8 ± 1.3	57.4 ± 4.6	59.6 ± 6.2	31.0 ± 1.8	49.2 ± 6.8	16.2 ± 1.4
10	17	76.8 ± 1.1	60.3 ± 0.6	75.1 ± 0.7	63.0 ± 1.3	79.6 ± 1.4	48.5 ± 1.4
10	18	31.3 ± 1.1	0.0 ± 0.5	20.7 ± 1.5	23.0 ± 9.0	8.5 ± 1.9	12.5 ± 1.5
10	19	1.8 ± 3.4	11.5 ± 0.0	19.7 ± 2.5	7.1 ± 1.2	−9.7 ± 3.3	9.8 ± 5.9
10	20	8.0 ± 3.5	14.7 ± 0.7	7.9 ± 4.7	5.4 ± 3.5	−13.5 ± 2.6	9.4 ± 2.2
10	21	7.8 ± 4.7	2.7 ± 1.6	13.1 ± 5.0	4.8 ± 1.8	−18.9 ± 10.1	5.8 ± 1.4
10	22	37.1 ± 2.4	25.8 ± 2.1	40.3 ± 1.1	26.4 ± 2.2	14.5 ± 2.2	25.0 ± 3.4
10	23	70.5 ± 1.3	54.1 ± 2.7	81.1 ± 3.1	58.6 ± 3.1	36.8 ± 11.3	42.4 ± 2.5
50	pyrimethanil	84.0 ± 1.3	65.9 ± 1.1	75.2 ± 14.3	39.9 ± 2.8	100.0 ± 0.0	26.1 ± 1.6
10	azoxystrobin	58.4 ± 0.6	46.0 ± 1.3	54.2 ± 2.1	47.6 ± 3.4	- c	39.0 ± 0.8

Note: ^a: concentration. ^b: R. s, Rhizoctonia solani; P. o, Pyricularia oryzae; C. m, Colletotrichum musae; F. g, Fusarium graminearum; B. c, Botrytis cinerea; and C. s, Colletotrichum siamense. ^c: Not tested.

. Subsequent evaluations of compounds with superior activity determined their median effective concentrations (EC₅₀), as detailed in

Table 2. EC₅₀ ^a values of some target compounds against six pathogenic fungi (µg/mL).

Compound	R s b	P. o	C. m	F. g	B. c	C. s
1	8.08	24.06	17.27	31.70	21.96	- c
16	7.28	14.98	12.08	22.14	4.17	52.19
17	2.88	5.96	4.32	6.23	2.99	9.09
23	4.24	8.92	3.20	44.40	18.43	14.33
azoxystrobin	<0.01	12.21	5.09	16.54	-	31.20

Note: ^a: The detailed toxicity equation is presented in Table S1. ^b: R. s, Rhizoctonia solani; P. o, Pyricularia oryzae; C. m, Colletotrichum musae; F. g, Fusarium graminearum; B. c, Botrytis cinerea; and C. s, Colletotrichum siamense. ^c: Not tested.

. The data indicate a notable enhancement in antifungal activity for compounds 1, 3, 5, 7, and 9 compared to compounds 2, 4, 6, 8, and 10, suggesting that the presence of a connecting arm with a single oxygen atom positively influences antifungal efficacy. The antifungal activity of compounds 1 and 9 is comparable, and both are more effective than compounds 3, 5, and 7, indicating that the concurrent presence of ether bonds and trifluoromethyl groups at the second and third positions of the pyridine ring in the compound is beneficial for enhancing its antifungal activity. Additionally, the activity of 1 and 12 surpasses that of 11, and compound 17 shows superior activity over 16, further confirming that chlorine atom substitution on the benzene ring is beneficial for augmenting antifungal activity. As indicated in Table 2, compound 17 (EC₅₀ 2.88~9.09 µg/mL) outperforms both 1 (EC₅₀ 8.08~21.96 µg/mL) and 16 (EC₅₀ 4.17~52.19 µg/mL) in terms of antifungal activity. The aromatic amino groups in 17 were subjected to amidation, leading to the synthesis of compounds 18~23. Unfortunately, those compounds largely lost their antifungal properties, with only compound 23 maintaining activity comparable to 17.

To further elucidate the antifungal activity of the compounds, we evaluated the in vivo protective capacity of compounds 17 and 23 against Colletotrichum musae. The data are listed in

Table 3. In vivo protective effect of compounds against Colletotrichum musae (%).

Compound	Concentration (μg/mL)	Protective Effect
		Disease Index	Control Efficacy (%)
17	200	46.67 ± 1.56 b	46.24 ± 2.34 b
23	200	42.22 ± 0.91 b	48.45 ± 1.46 b
azoxystrobin	200	24.44 ± 0.32 c	70.27 ± 0.67 a
CK	-	82.22 ± 0.54 a	-

Note: Lowercase letters represent one-way ANOVA results between different groups at the same time point (Duncan analysis p ≤ 0.05). a, b and c indicate significant differences between different groups, and a, b, and c are labeled in descending order of inhibition rate.

. At a concentration of 200 μg/mL, the protective effects of compounds 17 and 23 against C. musae were nearly comparable, with disease control rates of 46.24% and 48.45%, respectively. These rates are inferior to the in vivo activity exhibited by the positive control compound, azoxystrobin, suggesting that there is scope for enhancing the antifungal potency of these compounds.

2.3. Degradation of Compounds 17 and 23 in Bananas

In our preliminary studies, we found that compound A is photochemically unstable owing to the presence of an α, β-unsaturated ketone, which undergoes complete degradation upon 32 h of light exposure [15]. Despite its potent in vitro antifungal activity, the compound’s efficacy in vivo is significantly reduced. To address this instability, we utilized a scaffold hopping strategy to convert the unsaturated ketone into a pyridine ring, thereby generating a more stable compound. The half-lives of compounds 17 and 23 in bananas were quantified. During the experiment, compounds 17 and 23 were detected exclusively in the peel of the bananas, with no detection in the fruit flesh. The results are presented in Figure 3 (detailed data in Table S2). Compound 17 exhibits a higher degradation rate within the initial 24 h, after which the degradation rate levels off, yielding a half-life of 176.9 h. In contrast, compound 23 degrades more rapidly during the first three days, followed by a plateau in degradation rate, with a half-life of 94.8 h. These results demonstrate that the modification has markedly enhanced the stability of the compounds.

2.4. Molecular Docking

Compound structure dictates properties, and compounds with similar structures tend to exhibit similar biological activities and target the same potential sites of action [30,31]. Pyrazole-4-carboxamide is a key functional group known to inhibit succinate dehydrogenase (Figure S70) [20,25], and compound 23 incorporates this group, implying a potential for targeting succinate dehydrogenase. To explore this possibility, we conducted molecular docking experiments to assess the binding interaction between compound 23 and succinate dehydrogenase. We selected thifluzamide, a fungicide known to inhibit succinate dehydrogenase, as the positive control because its structure is similar to that of compound 23. The molecular docking results are presented in Figure 4 and Figure 5. The succinate dehydrogenase A chain is shown in green, the B chain in cyan, the C chain in light magenta, and the D chain in orange. Compound 23 binds to succinate dehydrogenase with a binding energy of −7.03 kcal/mol, forming hydrogen bonds with the 39th SER amino acid residue of the C chain and the 173rd TRP amino acid residue of the B chain (Figure 4). Thifluzamide binds to succinate dehydrogenase with a binding energy of −6.98 kcal/mol, also forming a hydrogen bond with the 173rd TRP amino acid residue of the B chain (Figure 5). The binding energies of both compounds are comparable. As observed, both compounds share similar binding sites, and Figure 6 provides an overlaid view to facilitate comparison. These results indicate that the target of compound 23 may also be succinate dehydrogenase. Although the above results generated by virtual molecular docking may not perfectly reflect real-world situations, they still offer valuable insights for the molecular optimization of potential inhibitors.

3. Materials and Methods

3.1. General

Reagents were purchased from commercial sources and used as received. All anhydrous solvents used for synthesis were dried and purified using standard techniques prior to use. The melting points of the synthesized compounds were tested using an X-4 binocular microscope (Beijing Tech Instruments Co., Ltd., Beijing, China). ¹H NMR and ¹³C NMR spectra were obtained using a Bruker AV 400 spectrometer (Bruker Corp., Fallanden, Switzerland) in CDCl₃ or DMSO-d₆ solutions with tetramethylsilane (TMS) as the internal standard. The progress of the reaction was monitored by thin-layer chromatography on silica gel GF-254 and detected by UV. Column chromatography was performed using 200–300 mesh silica gel (Qingdao Marine Chemical Co., Ltd., Qingdao, China). High-resolution mass spectra were obtained with an Agilent 6210 ESI/TOF MS (Agilent Technologies, Inc., Santa Clara, CA, USA.). High-performance liquid chromatography (HPLC) analyses were conducted using an Agilent 1260 Infinity III instrument (Agilent Technologies, Tokyo, Japan).

3.2. Synthesis

General synthesis procedures for title compounds 1~17 [32].

A mixture of N,N-dimethylformamide (DMF, 8 mL) and 2,4-dichlorophenol (10 mmol), 3-trifluoromethyl-2-fluoropyridine (10 mmol), and anhydrous K₂CO₃ (15 mmol) was stirred and refluxed for 12 h. The reaction progress was monitored by TLC until completion. After completion of the reaction, the reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (30 mL × 3). The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel using a petroleum ether (PE)/ethyl acetate (EA) gradient to obtain the target compounds.

2-(2,4-Dichlorophenoxy)-3-(trifluoromethyl) pyridine (1): Rf 0.5 (PE/EA 2:1), White solid, yield 76%, mp 45–46 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.25 (ddd, J = 4.9, 1.9, 0.8 Hz, 1H, 4-H-Py), 8.02 (ddd, J = 7.6, 1.8, 0.8 Hz, 1H, 6-H-Py), 7.49 (d, J = 2.5 Hz, 1H, 3-H-Ph), 7.32 (dd, J = 8.6, 2.5 Hz, 1H, 5-H-Ph), 7.19 (d, J = 8.6 Hz, 1H, 6-H-Ph), 7.12 (ddd, J = 7.6, 5.0, 0.9 Hz, 1H, 5-H-Py). ¹³C NMR (100 MHz, CDCl₃) δ 159.3 (q, J = 1.8 Hz), 150.7, 147.7, 137.3 (q, J = 4.7 Hz), 131.6, 130.4, 128.4, 128.1, 125.0, 122.6 (q, J = 272.1 Hz), 118.3, 113.8 (q, J = 34.0 Hz). HRMS (ESI) m/z calculated for C₁₂H₇NOF₃Cl₂⁺ [M + H]⁺, 307.9851; found, 307.9855.

2-((2,4-Dichlorobenzyl)oxy)-3-(trifluoromethyl) pyridine (2): Rf 0.45 (PE/EA 2:1), White solid, yield 71%, mp 74–75 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (dd, J = 5.1, 1.8 Hz, 1H, 4-H-Py), 7.90 (dd, J = 7.6, 1.9 Hz, 1H, 6-H-Py), 7.52 (d, J = 8.3 Hz, 1H, 6-H-Ph), 7.40 (d, J = 2.1 Hz, 1H, 3-H-Ph), 7.26 (dd, J = 8.2, 2.0 Hz, 1H, 5-H-Ph), 7.01 (dd, J = 7.5, 5.0 Hz, 1H, 5-H-Py), 5.55 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 159.7 (q, J = 1.7 Hz), 150.5, 136.6 (q, J = 4.8 Hz), 133.9, 133.2, 129.3, 129.1, 127.3, 123.0 (q, J = 271.8 Hz), 116.6, 113.4 (q, J = 33.2 Hz), 64.72. HRMS (ESI) m/z calculated for C₁₃H₉NOF₃Cl₂⁺ [M + H]⁺, 322.0008; found, 322.0014.

2-(2,4-Dichlorophenoxy)-5-(trifluoromethyl)pyridine (3): Rf 0.45 (PE/EA 2:1), Light yellow liquid, yield 68%. ¹H NMR (400 MHz, CDCl₃) δ 8.41–8.36 (m, 1H, 6-H-Py), 7.94 (dd, J = 8.6, 2.5 Hz, 1H, 4-H-Py), 7.50 (d, J = 2.5 Hz, 1H, 3-H-Ph), 7.32 (dd, J = 8.7, 2.5 Hz, 1H, 5-H-Ph), 7.17 (d, J = 8.6 Hz, 1H, 6-H-Ph), 7.12 (dt, J = 8.7, 0.7 Hz, 1H, 3-H-Py). ¹³C NMR (100 MHz, CDCl₃) δ 164.6, 147.8, 145.3 (q, J = 4.4 Hz), 137.0 (q, J = 3.3 Hz), 131.7, 130.5, 128.3, 128.2, 124.8, 123.6 (q, J = 270.0 Hz), 122.1 (q, J = 33.0 Hz), 111.2. HRMS (ESI) m/z calculated for C₁₂H₇NOF₃Cl₂⁺ [M + H]⁺, 307.9851; found, 307.9860.

2-((2,4-Dichlorobenzyl)oxy)-5-(trifluoromethyl) pyridine (4): Rf 0.45 (PE/EA 2:1), White solid, yield 67%, mp 63–64 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.46 (dt, J = 2.8, 1.0 Hz, 1H, 6-H-Py), 7.81 (dd, J = 8.7, 2.5 Hz, 1H, 4-H-Py), 7.46 (d, J = 8.3 Hz, 1H, 6-H-Ph), 7.43 (d, J = 2.1 Hz, 1H, 3-H-Ph), 7.26 (dd, J = 8.3, 2.1 Hz, 1H, 5-H-Ph), 6.91 (dt, J = 8.6, 0.8 Hz, 1H, 3-H-Py), 5.49 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 145.0 (q, J = 4.4 Hz), 136.0 (q, J = 3.2 Hz), 134.4, 134.1, 133.0, 130.4, 129.4, 127.2, 123.9 (q, J = 270.8 Hz), 120.6 (q, J = 33.1 Hz), 111.3, 65.0. HRMS (ESI) m/z calculated for C₁₃H₉NOF₃Cl₂⁺ [M + H]⁺, 322.0008; found, 322.0016.

2-(2,4-Dichlorophenoxy)-6-(trifluoromethyl) pyridine (5): Rf 0.5 (PE/EA 2:1), colorless liquid, yield 68%. ¹H NMR (400 MHz, CDCl₃) δ 7.91–7.83 (m, 1H, 4-H-Py), 7.48 (d, J = 2.4 Hz, 1H, 3-H-Ph), 7.40 (d, J = 7.4 Hz, 1H, 3-H-Py), 7.29 (dd, J = 8.7, 2.5 Hz, 1H, 5-H-Ph), 7.20 (d, J = 8.7 Hz, 1H, 6-H-Ph), 7.15 (d, J = 8.3 Hz, 1H, 5-H-Py). ¹³C NMR (100 MHz, CDCl₃) δ 162.4, 147.9, 146.1 (q, J = 35.5 Hz), 140.8, 131.2, 130.4, 128.1, 128.1, 124.7, 120.9 (q, J = 274.0 Hz), 115.4 (q, J = 3.1 Hz), 114.4 (q, J = 1.2 Hz). HRMS (ESI) m/z calculated for C₁₂H₇NOF₃Cl₂⁺ [M + H]⁺, 307.9851; found, 307.9858.

2-((2,4-Dichlorobenzyl)oxy)-6-(trifluoromethyl) pyridine (6): Rf 0.55 (PE/EA 2:1), White solid, yield 74%, mp 54–55 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (t, J = 7.9 Hz, 1H, 4-H-Py), 7.53 (d, J = 8.3 Hz, 1H, 6-H-Ph), 7.42 (d, J = 2.1 Hz, 1H, 3-H-Ph), 7.33–7.22 (m, 2H, 5-H-Ph and 3-H-Py), 6.98 (d, J = 8.4 Hz, 1H, 5-H-Py), 5.49 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 163.1, 145.5 (q, J = 34.7 Hz), 139.7, 134.8, 134.6, 133.0, 131.5, 129.4, 127.1, 121.3 (q, J = 274.8 Hz), 114.8 (q, J = 1.2 Hz), 113.8 (q, J = 3.2 Hz), 64.9. HRMS (ESI) m/z calculated for C₁₃H₉NOF₃Cl₂⁺ [M + H]⁺, 322.0008; found, 322.0014.

3-Chloro-2-(2,4-dichlorophenoxy)-5-(trifluoromethyl) pyridine (7): Rf 0.5 (PE/EA 2:1), colorless liquid, yield 77%. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (dt, J = 2.2, 1.0 Hz, 1H, 4-H-Py), 8.01 (d, J = 2.2 Hz, 1H, 6-H-Py), 7.51 (d, J = 2.5 Hz, 1H, 3-H-Ph), 7.34 (dd, J = 8.7, 2.5 Hz, 1H, 5-H-Ph), 7.19 (d, J = 8.7 Hz, 1H, 6-H-Ph). ¹³C NMR (100 MHz, CDCl₃) δ 160.1, 147.6, 142.5 (q, J = 4.4 Hz), 136.7 (q, J = 3.4 Hz), 132.1, 130.5, 128.3, 128.2, 124.8, 123.1 (q, J = 33.8 Hz), 122.7 (q, J = 272.3 Hz), 119.0. HRMS (ESI) m/z calculated for C₁₂H₆NOF₃Cl₃⁺ [M + H]⁺, 341.9462; found, 341.9470.

3-Chloro-2-((2,4-dichlorobenzyl)oxy)-5-(trifluoromethyl) pyridine (8): Rf 0.5 (PE/EA 2:1), White solid, yield 65%, mp 138–139 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (dq, J = 2.6, 1.3 Hz, 1H, 4-H-Py), 7.67 (d, J = 2.5 Hz, 1H, 3-H-Ph), 7.50–7.44 (m, 2H, 5-H-Ph and 6-H-Py), 7.29–7.26 (m, 1H, 6-H-Ph), 5.26 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 158.5, 135.7, 135.5 (q, J = 5.4 Hz), 134.5, 133.3 (q, J = 2.5 Hz), 132.7, 130.4, 129.8, 127.9, 127.8, 122.6 (q, J = 269.7 Hz), 109.5 (q, J = 35.8 Hz), 51.4. HRMS (ESI) m/z calculated for C₁₃H₇NOF₃Cl₃Na⁺ [M + Na]⁺, 377.9438; found, 377.9442.

3-(2,4-Dichlorophenoxy)-2-(trifluoromethyl) pyridine (9): Rf 0.5 (PE/EA 2:1), Dark yellow liquid, yield 72%. ¹H NMR (400 MHz, CDCl₃) δ 8.43 (dd, J = 4.7, 1.2 Hz, 1H, 6-H-Py), 7.53 (d, J = 2.5 Hz, 1H, 3-H-Ph), 7.43 (dd, J = 8.5, 4.6 Hz, 1H, 5-H-Py), 7.30 (dd, J = 8.7, 2.5 Hz, 1H, 5-H-Ph), 7.11 (dd, J = 8.5, 1.2 Hz, 1H, 4-H-Py), 7.04 (d, J = 8.7 Hz, 1H, 6-H-Ph). ¹³C NMR (100 MHz, CDCl₃) δ 151.9, 149.0, 143.2, 137.8 (q, J = 34.7 Hz), 131.6, 131.0, 128. 7, 127.7, 127.6, 124.7, 122.9, 121.4 (q, J = 274.8 Hz). HRMS (ESI) m/z calculated for C₁₂H₇NOF₃Cl₂⁺ [M + H]⁺, 307.9851; found, 307.9858.

3-((2,4-Dichlorobenzyl)oxy)-2-(trifluoromethyl) pyridine (10): Rf 0.5 (PE/EA 2:1), White solid, yield 68%, mp 117–118 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.32 (dd, J = 4.5, 1.3 Hz, 1H, 6-H-Py), 7.55 (dd, J = 8.4, 1.0 Hz, 1H, 4-H-Py), 7.48 (dd, J = 8.5, 4.5 Hz, 1H, 5-H-Py), 7.45–7.40 (m, 2H, 3-H-Ph and 6-H-Ph), 7.33 (dd, J = 8.4, 2.1 Hz, 1H, 5-H-Ph), 5.25 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 152.8, 141.0, 137.1 (q, J = 34.09 Hz), 134.6, 132.6, 131.8, 129.3, 129.1, 127.8, 127.7, 121.7 (q, J = 274.5 Hz), 121.2, 67.0. HRMS (ESI) m/z calculated for C₁₃H₉NOF₃Cl₂⁺ [M + H]⁺, 322.0008; found, 322.0015.

2-(2,4-Dimethylphenoxy)-3-(trifluoromethyl) pyridine (11): Rf 0.5 (PE/EA 2:1), Pale yellow solid, yield 70%, mp 62–63 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (ddd, J = 5.0, 1.8, 0.8 Hz, 1H, 4-H-Py), 7.97 (ddd, J = 7.5, 1.9, 0.8 Hz, 1H, 6-H-Py), 7.11–7.08 (m, 1H, 5-H-Py), 7.07–7.01 (m, 2H, 3-H-Ph and 5-H-Ph), 6.98 (d, J = 8.1 Hz, 1H, 6-H-Ph), 2.34 (s, 3H, CH₃), 2.11 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 160.4, 151.0, 149.0, 137.0 (q, J = 4.7 Hz), 135.3, 132.0, 130.3, 127.6, 123.0 (q, J = 271.9 Hz), 121.9, 117.1, 113.6 (q, J = 33.4 Hz), 20.9, 16.2. HRMS (ESI) m/z calculated for C₁₄H₁₃NOF₃⁺ [M + H]⁺, 268.0944, found; 268.0945.

2-(2,6-Dichlorophenoxy)-3-(trifluoromethyl) pyridine (12): Rf 0.5 (PE/EA 2:1), White solid, yield 51%, mp 96–97 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (ddd, J = 5.0, 1.8, 0.8 Hz, 1H, 4-H-Py), 8.03 (ddd, J = 7.5, 1.8, 0.8 Hz, 1H, 6-H-Py), 7.40 (d, J = 8.1 Hz, 2H, 3-H-Ph and 5-H-Ph), 7.18 (dd, J = 8.5, 7.8 Hz, 1H, 4-H-Ph), 7.13 (ddt, J = 7.5, 5.0, 0.8 Hz, 1H, 5-H-Py). ¹³C NMR (100 MHz, CDCl₃) δ 158.40 (q, J = 1.7 Hz), 150.7, 145.6, 137.31 (q, J = 4.7 Hz), 129.6, 128.8, 126.9, 122.7 (q, J = 272.2 Hz), 118.3, 113.5 (q, J = 34.2 Hz). HRMS (ESI) m/z calculated for C₁₂H₇NOF₃Cl₂⁺ [M + H]⁺, 307.9851; found, 307.9854.

2-((3-(Trifluoromethyl)pyridin-2-yl)oxy)aniline (13): Rf 0.5 (PE/EA 1:1), Light pink solid, yield 84%, mp 73–74 °C. ¹H NMR (400 MHz, CDCl₃) δ (dd, J = 5.1, 1.8 Hz, 1H, 4-H-Pyr), 7.99 (dd, J = 7.5, 1.9 Hz, 1H, 6-H-Py), 7.10 (d, J = 1.5 Hz, 1H, Ph), 7.09–7.06 (m, 2H, Ph), 6.87 (dd, J = 8.3, 1.6 Hz, 1H, Ph), 6.85–6.79 (m, 1H, 5-H-Py), 3.37 (br, 2H, NH₂). ¹³C NMR (100 MHz, CDCl₃) δ 159.9 (q, J = 1.9 Hz), 151.2, 140.1, 138.8, 137.0 (q, J = 4.7 Hz), 126.5, 123.0 (q, J = 272.0 Hz), 122.82, 118.9, 117.8, 117.0, 113.9 (q, J = 33.1 Hz). HRMS (ESI) m/z calculated for C₁₂H₁₀N₂OF₃⁺ [M + H]⁺, 255.0740; found, 255.0743.

3-((3-(Trifluoromethyl)pyridin-2-yl)oxy)aniline (14): Rf 0.5 (PE/EA 1:1), White solid, yield 79%, mp 75–76 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (ddd, J = 5.0, 1.9, 0.8 Hz, 1H, 4-H-Py), 7.97 (ddd, J = 7.5, 1.9, 0.8 Hz, 1H, 6-H-Py), 7.18 (t, J = 8.0 Hz, 1H, Ph), 7.07 (ddd, J = 7.6, 4.9, 0.8 Hz, 1H, 5-H-Py), 6.58–6.51 (m, 2H, Ph), 6.49 (t, J = 2.2 Hz, 1H, Ph), 3.37 (s, 2H, NH₂). ¹³C NMR (100 MHz, CDCl₃) δ 160.5, 154.2, 151.0, 147.8, 137.0 (q, J = 4.7 Hz), 130.2, 122.8 (q, J = 271.9 Hz), 117.6, 114.4 (q, J = 33.4 Hz), 112.3, 111.6, 108.4. HRMS (ESI) m/z calculated for C₁₂H₁₀N₂OF₃⁺ [M + H]⁺, 255.0740; found, 255.0740.

4-((3-(Trifluoromethyl)pyridin-2-yl)oxy)benzoic acid (15): Rf 0.45 (PE/EA 1:1), White solid, yield 43%, mp 157–158 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (dd, J = 5.0, 1.8 Hz, 1H, 4-H-Py), 8.31 (dd, J = 7.7, 1.8 Hz, 1H, 6-H-Py), 8.01 (d, J = 7.8 Hz, 2H, 2-H-Ph and 6-H-Ph), 7.39 (dd, J = 7.7, 4.9 Hz, 1H, 5-H-Py), 7.27 (d, J = 7.0 Hz, 2H, 3-H-Ph and 5-H-Ph). ¹³C NMR (100 MHz, DMSO-d₆) δ 159.4, 1568, 152.2, 138.6 (q, J = 4.6 Hz), 131.5, 124.7, 122.0, 121.6, 120.0, 113.5 (q, J = 32.9 Hz). HRMS (ESI) m/z calculated for C₁₃H₇NO₃F₃⁻ [M-H]⁻, 282.0384; found, 282.0389.

3-Chloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)aniline (16): Rf 0.5 (PE/EA 2:1),Brick red solid, yield 63%, mp 113–114 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (ddd, J = 5.0, 1.9, 0.8 Hz, 1H, 4-H-Py), 7.98 (ddd, J = 7.6, 1.9, 0.8 Hz, 1H, 6-H-Py), 7.06 (ddd, J = 7.6, 5.0, 0.8 Hz, 1H, 5-H-Py), 7.02 (d, J = 8.6 Hz, 1H, 6- H-Ph), 6.78 (d, J = 2.7 Hz, 1H, 3- H-Ph), 6.61 (dd, J = 8.6, 2.7 Hz, 1H, 5- H-Ph), 3.5 (br, 2H, NH₂). ¹³C NMR (100 MHz, CDCl₃) δ 160.2, 150.8, 145.1, 140.7, 137.0 (q, J = 4.7 Hz), 127.7, 124.5, 122.8 (q, J = 271.9 Hz), 117.6, 116.4, 114.4, 113.6 (q, J = 33.8 Hz). HRMS (ESI) m/z calculated for C₁₂H₉N₂OF₃Cl⁺ [M + H]⁺, 289.0350; found, 289.0351.

3,5-Dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)aniline (17): Rf 0.5 (PE/EA 2:1), White solid, yield 56%, mp 142–143 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.25 (ddd, J = 5.0, 1.9, 0.8 Hz, 1H, 4-H-Py), 8.00 (ddd, J = 7.6, 1.9, 0.8 Hz, 1H, 6-H-Py), 7.10 (ddd, J = 7.6, 5.0, 0.8 Hz, 1H, 5-H-Py), 6.67 (s, 2H, Ph), 3.52 (s, 2H, NH₂) ¹³C NMR (100 MHz, CDCl₃) δ 159.0 (q, J = 1.6 Hz), 150.7, 145.1, 137.2 (q, J = 4.7 Hz), 137.0, 129.5, 122.7 (q, J = 272.2 Hz), 118.1, 114.8, 113.4 (q, J = 34.1 Hz). HRMS (ESI) m/z calculated for C₁₂H₈N₂OF₃Cl₂⁺ [M + H]⁺, 322.9960; found, 322.9965.

General Synthesis Procedures for Title Compounds 18~23.

Compound 17 (1 mmol) and Et₃N (4 mmol) were added to dry DCM (10 mL) and stirred, followed by the dropwise addition of an excess of acyl chloride (5 mmol) via a dropping funnel. The mixture was stirred at room temperature for 2 h, then diluted with additional DCM (20 mL) and washed with a saturated NaHCO₃ solution (5 mL). The organic layer was then evaporated under reduced pressure. The residue was purified by flash column chromatography on silica gel using a PE/EA gradient, affording compounds 18~23.

Isopropyl(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)carbamate (18): Rf 0.5 (PE/EA 2:1),Yellow solid, yield 73%, mp 127–128 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.03 (s, 1H, NH), 8.37 (dd, J = 5.0, 1.8 Hz, 1H, 4-H-Py), 8.33 (dd, J = 7.7, 1.8 Hz, 1H, 6-H-Py), 7.68 (s, 2H, Ph), 7.39 (dd, J = 7.6, 5.0 Hz, 1H, 5-H-Py), 4.92 (hept, J = 6.2 Hz, 1H, CH), 1.28 (d, J = 6.2 Hz, 6H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.1, 153.5, 151.9, 139.5, 134.0, 138.8 (q, J = 4.8 Hz), 128.7, 123.3 (q, J = 272.0 Hz), 120.0, 118.27, 112.0 (q, J = 33.7 Hz), 68.9, 22.3. HRMS (ESI) m/z calculated for C₁₆H₁₂N₂O₃F₃Cl₂⁻ [M-H]⁻, 407.0183; found, 407.0191.

N-(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)benzamide (19): Rf 0.45 (PE/EA 2:1), White solid, yield 78%, mp 175–176 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.27–8.20 (m, 1H, 4-H-Py), 8.07–7.99 (m, 1H, 6-H-Py), 7.90 (s, 1H, NH), 7.88–7.83 (m, 2H, Ph), 7.80 (s, 2H, Ph), 7.63–7.55 (m, 1H, Ph), 7.55–7.46 (m, 2H, Ph), 7.18–7.10 (m, 1H, 5-H-Py). ¹³C NMR (100 MHz, CDCl₃) δ 165.8, 158.5, 150.7, 141.9, 137.3 (q, J = 4.6 Hz), 136.4, 134.1, 132.4, 129.7, 129.0, 127.1, 122.7 (q, J = 272.1 Hz), 120.1, 118.4, 113.5 (q, J = 34.3 Hz). HRMS (ESI) m/z calculated for C₁₉H₁₁N₂O₂F₃Cl₂Na⁺ [M + Na]⁺, 449.0042; found, 449.0043.

N-(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)-2,4-difluorobenzamide (20): Rf 0.45 (PE/EA 2:1), Beige solid, yield 72%, mp 176–177 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.37 (d, J = 16.0 Hz, 1H, Ph), 8.27–8.17 (m, 2H, Ph and NH), 8.06–8.02 (m, 1H, Py), 7.80 (s, 2H, Ph), 7.17–7.12 (m, 1H, Py), 7.12–7.06 (m, 1H, Py), 6.97 (ddd, J = 12.3, 8.3, 2.4 Hz, 1H, Ph). ¹³C NMR (100 MHz, CDCl₃) δ 165.4 (dd, J = 257.3, 13.2 Hz), 160.8 (dd, J = 248.6, 12.4 Hz), 160.4 (d, J = 4.0 Hz), 158.5, 150.7, 142.2, 137.3 (q, J = 4.7 Hz), 135.9, 134.2 (dd, J = 10.4, 3.5 Hz), 129.8, 122.6 (q, J = 272.2 Hz) 120.5, 118.4, 117.1 (dd, J = 11.4, 3.8 Hz), 113.5 (q, J = 34.2 Hz), 113.1 (dd, J = 21.4, 3.2 Hz), 104.6 (dd, J = 29.1, 26.0 Hz). HRMS (ESI) m/z calculated for C₁₉H₉N₂O₂F₅Cl₂Na⁺ [M + Na]⁺, 484.9853; found, 484.9861.

N-(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)-3-(2,4-dichlorophenyl)acrylamide (21): Rf 0.45 (PE/EA 2:1), White solid, yield 61%, mp 250–251 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H, NH), 8.39 (dd, J = 5.0, 1.7 Hz, 1H, 4-H-Py), 8.35 (dd, J = 7.7, 1.8 Hz, 1H, 6-H-Py), 7.93 (s, 2H, Ph), 7.90–7.81 (m, 2H, CH and 6-H-Ph), 7.78 (d, J = 2.2 Hz, 1H, 3-H-Ph), 7.56 (dd, J = 8.5, 2.2 Hz, 1H, 5-H-Ph), 7.40 (dd, J = 7.6, 5.0 Hz, 1H, 5-H-Py), 6.85 (d, J = 15.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.8, 158.1, 151.9, 140.6, 138.9, 138.4, 135.8, 135.6, 134.9, 131.7, 130.1, 129.6, 128.8, 128.6, 125.5, 123.2 (q, J = 271.9 Hz), 120.1, 119.7, 112.1 (q, J = 33.9 Hz). HRMS (ESI) m/z calculated for C₂₁H₁₁N₂O₂F₃Cl₄Na⁺ [M + Na]⁺, 542.9419; found, 542.9423.

N-(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)-2-phenylacetamide (22): Rf 0.45 (PE/EA 2:1), Pale yellow solid, yield 48%, mp over 300 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (dd, J = 5.1, 1.8 Hz, 1H, 4-H-Py), 8.01 (ddd, J = 7.5, 1.8, 0.8 Hz, 1H, 6-H-Py), 7.55 (s, 2H, Ph), 7.46–7.40 (m, 2H, Ph), 7.40–7.34 (m, 1H, Ph), 7.34–7.30 (m, 2H, Ph), 7.12 (dd, J = 7.6, 5.0 Hz, 1H, 5-H-Py), 7.08 (s, 1H, NH), 3.75 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 169.2, 158.5, 150.6, 141.8, 137.3 (q, J = 4.6 Hz), 136.1, 133.8, 129.6, 129.5, 129.4, 128.0, 122.6 (q, J = 272.2 Hz), 119.6, 118.4, 113.5 (q, J = 34.3 Hz), 44.8. HRMS (ESI) m/z calculated for C₂₀H₁₂N₂O₂F₃Cl₂⁻ [M-H]⁻, 439.0233; found, 439.0217.

N-(3,5-dichloro-4-((3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (23): Rf 0.5 (PE/EA 2:1), Pale yellow solid, yield 76%, mp 158–159 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (dd, J = 5.1, 1.8 Hz, 1H, 4-H-Py), 8.18 (t, J = 5.8 Hz, 1H, H-Pyr), 8.05 (s, 1H, NH), 8.03 (dd, J = 7.4, 1.8 Hz, 1H, 6-H-Py), 7.74 (s, 2H, Ph), 7.13 (dd, J = 7.6, 5.0 Hz, 1H, 5-H-Py), 6.88 (t, J = 54.2 Hz, 1H, CH), 3.97 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 159.1, 158.5 (q, J = 1.6 Hz), 150.7, 142.4 (t, J = 29.3 Hz), 141.9, 137.3 (q, J = 4.8 Hz), 136.3, 136.2, 129.7, 122.7 (q, J = 272.0 Hz), 112.0, 118.4, 116.5, 113.5 (q, J = 34.3 Hz), 112.2 (t, J = 232.8 Hz), 39.7. HRMS (ESI) m/z calculated for C₁₈H₁₀N₄O₂F₅Cl₂⁻ [M-H]⁻, 479.0106; found, 479.0105.

3.3. Biological Assays

Antifungal effects of title compounds in vitro [33,34].

The antifungal activity of a series of target compounds was evaluated in vitro against six plant pathogenic fungi, Rhizoctonia solani, Pyricularia oryzae, Colletotrichum musae, Fusarium graminearum, Botrytis cinerea, and Colletotrichum siamense, using the mycelial growth rate method. The plant pathogens were collected from the field and identified with morphology and molecular phylogenetic analysis by Professor Changping Xie from the School of Tropical Agriculture and Forestry at Hainan University and stored in the Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education. All six of the plant pathogenic fungi had good infecting ability. The target compounds were prepared as a solution in DMF and then incorporated into the potato dextrose agar (PDA) medium, maintaining a consistent concentration of 10 or 50 μg/mL in the medium. Subsequently, 5 mm mycelial disks were placed at the center of the PDA plates (with three plates per treatment) and maintained at a temperature of 27 ± 1 °C for a period ranging from 4 to 7 days. The experimental setup was conducted in triplicate for each compound. DMF without compounds served as the negative control, while pyrimethanil and azoxystrobin were applied as the reference standards. Subsequently, the toxic regression equations and median effective concentrations (EC₅₀) of some active compounds were determined. The test compounds were diluted to five distinct concentrations, and the inhibition percentage for each concentration was quantified. The logarithmic transformation of these concentrations and their associated inhibition percentages were then employed to conduct a linear regression, which yielded the EC₅₀ value. The inhibition of the title compound against these fungi was calculated by the following formula:

$$\mathrm{I}={\displaystyle \frac{\mathrm{C}-\mathrm{T}}{\mathrm{C}-0.5}} \times 100$$

I represents the inhibition rate, C represents the diameter of fungal growth on untreated PDA, and T represents the diameter of fungi on treated PDA.

In vivo protective activity against C. musae on bananas [35].

The freshly purchased bananas were washed with water and disinfected with 75% ethanol, then rinsed with distilled water for later use. The compound was dissolved in 0.2 mL of dimethyl sulfoxide (DMSO) and diluted with 0.2% Triton-100 in water to a final concentration of 200 µg/mL test solution. This solution was evenly sprayed onto the bananas. After 24 h, a spore suspension of C. musae at a concentration of 5–6 × 10⁸ CFU/mL was applied to the bananas. Solvents were used as the negative control, and azoxystrobin served as the positive control. The treated bananas were then incubated at 25 °C and 80% relative humidity, and the incidence of disease was assessed 5–7 days later. The grading criteria are as follows: Grade 0: No disease spots. Level 1: The lesion area is less than 5% of the fruit area. Level 3: Disease spot area accounts for 6% to 10% of the fruit area. Level 5: Disease spot area accounts for 11% to 25% of the fruit area. Level 7: Disease spot area accounts for 26% to 50% of the fruit area. Level 9: The lesion area accounts for more than 50% of the fruit area.

$$\mathrm{Disease} \mathrm{index}={\displaystyle \frac{\sum \left(\mathrm{incidence} \mathrm{number} \mathrm{of} \mathrm{all} \mathrm{levels} \text{×} \mathrm{representative} \mathrm{value} \mathrm{of} \mathrm{this} \mathrm{level}\right)}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{investigated} \mathrm{leaves} \text{×} \mathrm{highest} \mathrm{value}}}\times 100$$

$$\mathrm{Control} \mathrm{effect}={\displaystyle \frac{\mathrm{control} \mathrm{disease} \mathrm{index} - \mathrm{treatment} \mathrm{disease} \mathrm{index}}{\mathrm{control} \mathrm{disease} \mathrm{index}}}\times 100\%$$

3.4. Degradation of Compounds 17 and 23 in Bananas [36]

Bananas of unblemished and uniform size, procured from the local market, were immersed in a solution containing 200 μg/mL of the test compound for a period of 30 min. The test compound was dissolved with DMSO and configured into 1000 μg/mL mother liquor, which was diluted to the required concentration with 0.5% Tween-80 water. The evaluation of each compound was conducted in triplicate. Following immersion, the peels and pulps of the bananas were collected at specified time intervals: 2 h, 4 h, 24 h, 72 h, and 120 h after exposure to the compound solution. A 10 g portion of the collected sample was homogenized using a tissue grinder. Subsequently, 50 mL of dichloromethane was added to the homogenate, followed by sonication and shaking for a duration of 30 min. The suspension was then filtered, and the filtrate was evaporated under reduced pressure. The residue was redissolved in chromatographic-grade methanol and diluted to a final volume of 10 mL. The solution filtered through a 0.22 μm organic phase filter membrane and was transferred into a sample vial for analysis.

Chromatographic conditions. The analysis was performed on a Cosmosil 5C18-MS-II column (5 μm, 4.6 × 250 mm) with the column temperature maintained at 25 °C. A sample volume of 10 μL was injected for analysis. The mobile phase comprised a mixture of 30% water and 70% methanol, and the flow rate was set to 1 mL/min.

Linear regression equation. Five distinct concentrations of standard solutions were prepared by diluting the original standard solution with methanol. The test compound injection concentration (x, μg/mL) was as the abscissa and the corresponding chromatographic peak area (y) as the ordinate for the linear regression calculation. The linear equation for compound 17 was y = 15.31x − 359.99 (R², 0.997) (Figure S72), and the linear equation for compound 23 was y = 14.922x + 160.09 (R², 0.9999) (Figure S74).

Data analysis. The dissipation of the test compound in bananas over time was evaluated using a first-order kinetic equation, and the dissipation dynamic equation and half-life were calculated using the following formula:

C_t = C₀e^−kt

t_1/2 = ln2/k = 0.693/k

where C_t represents the residue concentration (mg/kg) at time t, C₀ is the initial concentration (mg/kg), k is the dissipation coefficient, and t_1/2 is the time required for test compound residue level to decrease to half of the initial residue concentration. Statistical analysis was performed using OriginPro 2024 SR1 10.1.0.178.

3.5. Molecular Docking Study

The crystal structure of the target protein, succinate dehydrogenase (SDH) (PDB code: 2FBW) [37], retrieved from the RCSB Protein Data Bank (https://www.rcsb.org, accessed on 15 February 2025), had its non-protein components removed using Discovery Studio Visualizer (version 24.1.0, Build 23298, developed by Dassault Systèmes, Vélizy-Villacoublay, France). Molecular docking experiments were conducted using AutoDockTools (version 1.5.7, The Scripps Research Institute, San Diego, CA, USA), adhering to the methodology outlined by Olson [38]. The docking procedures were carried out via the AutoDock algorithm. For global docking, a grid was used that encompassed the binding region between the B chain and the C and D chains of succinate dehydrogenase, with detailed information depicted in Figure S75 of the Supporting Information. The default settings were maintained for the majority of the docking parameters, with the exception of the following adjustment: the ‘Number of GA Runs’ was increased to 100. Visualization of the docking outcomes was achieved with PyMOL (version 3.1.3, provided by Schrödinger, New York, NY, USA), and the 2D diagrams illustrating ligand interactions were created using Discovery Studio Visualizer.

4. Conclusions

In this study, we employed a scaffold hopping approach to transform the α, β-unsaturated ketones of the antifungal lead compound dehydrozingerone into pyridine derivatives, resulting in the design and synthesis of a series of structurally novel trifluoromethylpyridine compounds. Our evaluation of their antifungal properties revealed that most compounds have good in vitro antifungal activity, especially in inhibiting Rhizoctonia solani and Colletotrichum musae. Compound 17 exhibited the most efficient and broad-spectrum in vitro antifungal activity, with EC₅₀ values ranging from 2.88 to 9.09 µg/mL. These phenoxytrifluoromethylpyridine derivatives exhibit superior antifungal efficacy compared to the benzyloxytrifluoromethylpyridine analogs. The antifungal activity is notably enhanced when the trifluoromethyl and ether groups are positioned on the same side of the nitrogen atom in the pyridine ring. Furthermore, the introduction of chlorine substitutions on the phenyl ring contributes to increased activity. Compound 17 in particular demonstrates broad-spectrum antifungal activity and is a promising candidate for further development as a lead compound. Both compounds 17 and 23 exhibited moderate in vivo control effects against C. musae. A degradation study of compounds 17 and 23 in bananas was conducted, and it was found that their half-lives were 176.9 and 94.8 h, respectively, indicating the stability of their structures in the environment. Molecular docking studies revealed that compound 23 interacts with succinate dehydrogenase with a binding energy of −7.03 kcal/mol, forming hydrogen bonds with the 39th SER residue of chain C and the 173rd TRP residue of chain B. These strong interactions provide valuable insights for the structural optimization of compound 23.