English

• 1.   Introduction

  Over the past few decades, plant parasitic nematodes have caused serious damage to crop production worldwide and have recently caused even more severe problems, ^{[1, 2]} however, the varieties of nematicides used on the market are deficient. Traditional highly toxic or virulent nematocides, such as carbamates aldicarb, carbofuran, oxamyl, and organophosphates fenamiphos, cadusafos, fensulfothion, etc., have been banned or restricted in China.^{[3, 4]} Furthermore, the early fumigant methyl bromide was also phased out due to its destruction of the ozone layer.^[4] Nowadays, the chemical control agents widely used in the market are mainly fosthiazate and avermectin B2a (Figure 1), whose long-term use has produced serious resistance.

  Figure 1

  

  Figure 1.  Chemical structures of fosthiazate, avermectin B2a and fluopyram

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  The discovery and development of new nematicides which are highly effective against the target pest, work by new modes of action, and meet societal demands of safety to humans and the environment are essential in the defense of crops.^[5] Fluopyram (Figure 1), a SDHI fungicide successfully developed by Bayer AG, has also been extensively used to control soil nematodes as a new generation of highly effective nematicide.^[6~8] Subsequently, some nematocidal amide structures were reported successively (Figure 2).^[9~15]

  Figure 2

  

  Figure 2.  Structures of reported nematocidal amide compounds

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  In our previous work, based on the recently reported nematocidal active ingredients, two series of amide derivatives were designed and synthesized by introducing sulfide and sulfone groups into the commercial fluopyram, most of which displayed excellent nematocidal activity at 200 μg/mL (Figure 3).^[16] In order to explore the effect of structural modification of the amide bridge on biological activity in a more delicate way, another two series of novel amide target compounds were designed and prepared by adopting amide group flipping and introducing N-sulfonyl substituted amide bonds (Figure 3).

  Figure 3

  

  Figure 3.  Design strategy of the target compounds

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  2.   Results and discussion

  2.1   Synthetic chemistry

  Herein, the important intermediates 2, 5 and the target compounds Ⅰ-1~Ⅰ-12, Ⅱ-1~Ⅱ-6 were designed and synthesized according to the methods in Scheme 1. Firstly, amides 2 were prepared by acylation of aromatic amines and chloroacetyl chloride. Given the weak nucleophilic property of sulfonamides, a catalytic amount of 4-dimethyl- aminopyridine (DMAP) was adopted to improve the reaction of aromatic sulfonamides and chloroacetyl chloride to provide intermediates 5. Aromatic thiophenols 3 were obtained by purchasing market varieties or laboratory preparation, of which 3-chloro-5-(trifluoromethyl)pyridine-2- thiol (3a) was synthesized by nucleophilic substitution of 2, 3-dichloro-5-(trifluoromethyl)pyridine and sodium hydrosulfide. Finally, intermediates 2, 5 and 3 were reacted respectively to generate eighteen novel amide target compounds Ⅰ-1~Ⅰ-12 and Ⅱ-1~Ⅱ-6, whose structures were identified by ¹H NMR, ¹³C NMR and HRMS. Unfortunately, the target compounds containing 5-methoxy-3H- imidazo[4, 5-b]pyridin-2-yl group in compounds Ⅱ were unavailable to obtain through the procedures described above.

  Scheme 1

  

  Scheme 1.  Synthetic routes of the target compounds

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  2.2   Biological activity

  The nematocidal activities of target compounds against M. incognita, with fluopyram as a positive control, were shown in Table 1. From the data, the target compounds Ⅰ-1~Ⅰ-12 showed extremely weak nematocidal activities, however compounds Ⅱ exhibited significantly higher mortality than compounds Ⅰ. This result indicated that the introduction of the N-sulfonyl substituent on the amide group was favorable to improving the nematocidal activity in comparison with compounds Ⅰ. Compared the target compounds Ⅰ-1~Ⅰ-12 with our previous work, it can be concluded that the position of the amide bonds had a great influence on their nematocidal activity, of which directly attaching the carbonyl group to the aromatic heterocyclic ring was advantageous. In addition, there was no significant difference in nematocidal activity among the target compounds Ⅱ containing different aromatic rings on both sides of the amide bridge, which suggested that N-sulfonyl amide group played an important role in nematocidal activity. Overall, compared to the control fluopyram, no compounds displayed excellent nematocidal activities at 200 and 100 μg/mL, whereas the bioassays of compounds Ⅱ-1~Ⅱ-6 would provide a valuable guide to further explore the potential, efficient nematocidal lead compounds.

  Table 1

  

  Table 1.  Nematocidal activity of the target compounds against M. incognita

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  Compound	Mortality/%
  	200 μg/mL	100 μg/mL
  Ⅰ-1	0	0
  Ⅰ-2	0	0
  Ⅰ-3	0	0
  Ⅰ-4	1.9±0.07	0
  Ⅰ-5	0	0
  Ⅰ-6	1.22±0.08	0
  Ⅰ-7	0	0
  Ⅰ-8	0	0
  Ⅰ-9	0	0
  Fluopyram	99.3±1.14	99.4±1.12
  Ⅰ-10	1.66±0.31	1.36±0.11
  Ⅰ-11	4.44±0.81	0
  Ⅰ-12	2.72±0.93	1.71±0.58
  Ⅱ-1	62.7±1.95	57.0±1.34
  Ⅱ-2	66.5±1.75	40.4±1.04
  Ⅱ-3	57.5±1.47	49.7±0.17
  Ⅱ-4	77.5±0.97	57.2±1.43
  Ⅱ-5	50.5±2.09	48.9±2.31
  Ⅱ-6	50.3±0.56	49.6±1.36

  Considering nematocide fluopyram was also used as an excellent SDHI fungicide, the fungicidal inhibition rates of target compounds were further measured and the results were shown in Table 2. Based on the data, no compounds displayed superior and broad-spectrum antifungal activity against all tested agricultural pathogens. In a given category, most of the target compounds exhibited higher inhibitory rates against Cercospora circumscissa Sacc. than other tested pathogens. In terms of the structural characteristics, there is no significant difference in fungicidal activity between compounds Ⅰ and Ⅱ, and among the same series. As a whole, the structural modification of the amide bridge had a great influence on the fungicidal activity.

  Table 2

  

  Table 2.  Fungicidal activity of the target compounds at 50 μg/mL^a

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  Compound	Inhibition rate/%
  	GZ	RS	CC	PM	BC	PP	AK	CS
  Fluopyram	55.6±0.09	44.3±0.09	74.9±0.38	67.5±0.03	92.2±0.15	79.3±0.13	100.0	100.0
  Ⅰ-1	8.3±0.14	19.0±0.05	61.1±0.43	31.7±0.05	9.7±0.24	25.9±0.06	21.5±0.06	22.1±0.10
  Ⅰ-2	4.2±0.24	16.5±0.01	6.3±0.17	48.0±0.13	9.7±0.26	15.5±0.05	21.5±0.06	52.3±0.24
  Ⅰ-3	19.4±0.18	20.3±0.26	38.3±0.40	41.5±0.08	9.8±0.13	36.2±0.05	40.6±0.22	57.3±0.10
  Ⅰ-4	1.4±0.21	21.5±0.13	10.9±0.18	25.2±0.10	9.7±0.42	41.4±0.05	26.8±0.15	55.7±0.08
  Ⅰ-5	38.9±0.15	26.6±0.22	47.4±0.25	64.2±0.05	9.7±0.13	12.1±0.10	0	64.9±0.10
  Ⅰ-6	15.3±0.18	21.5±0.09	47.4±0.41	57.7±0.01	9.7±0.13	75.9±0.06	34.2±0.05	67.9±0.10
  Ⅰ-7	0	21.5±0.14	20.0±0.13	33.3±0.19	9.6±0.15	41.4±0.14	21.5±0.06	19.1±0.15
  Ⅰ-8	22.2±0.28	19.0±0.10	13.1±0.08	38.2±0.10	12.8±0.06	46.6±0.12	68.2±0.10	42.0±0.15
  Ⅰ-9	15.3±0.06	17.0±0.14	33.7±0.39	38.2±0.14	9.7±0.22	20.7±0.09	14.3±0.07	34.4±0.17
  Ⅰ-10	27.8±0.13	39.2±0.57	61.1±0.47	46.3±0.02	9.5±0.21	11.2±0.11	13.0±0.10	86.3±0.05
  Ⅰ-11	22.2±0.25	8.9±0.22	58.9±0.61	57.7±0.08	90.7±0.13	45.7±0.15	34.2±0.05	34.4±0.15
  Ⅰ-12	22.2±0.10	10.1±0.24	33.7±0.14	35.0±0.25	75.1±0.06	43.1±0.10	63.9±0.10	84.7±0.06
  Ⅱ-1	13.9±0.31	8.9±0.02	7.4±0.06	57.7±0.30	7.2±0.07	25.9±0.17	25.7±0.13	26.7±0.10
  Ⅱ-2	22.2±0.27	20.3±0.08	40.6±0.31	28.5±0.22	5.1±0.08	19.0±0.09	8.8±0.19	69.5±0.13
  Ⅱ-3	22.2±0.12	11.4±0.10	81.7±0.33	44.7±0.01	25.3±0.06	70.7±0.10	51.2±0.05	52.7±0.06
  Ⅱ-4	23.6±0.45	0	24.6±0.22	25.2±0.10	29.2±0.13	8.6±0.21	8.8±0.17	86.3±0.10
  Ⅱ-5	6.9±0.11	16.5±0.13	13.1±0.13	51.2±0.01	40.1±0.15	29.3±0.17	30.0±0.10	52.7±0.10
  Ⅱ-6	9.7±0.20	13.9±0.17	7.4±0.15	44.7±0.19	26.8±0.08	55.2±0.13	70.3±0.73	53.2±0.15
  a GZ: Gibberella zeae; RS: Rhizoctonia solani; CC: Colletotrichum capsici; PM: Phyllosticta melongenae Sawada; BC: Botrytis cinerea; PP: Physalospora piricola; AK: Alternaria kikuchiana Tanaka; CS: Cercospora circumscissa Sacc.

  2.3   Molecular docking

  Although the composite crystal structure of fluopyram and the target enzyme SDH in nematodes has not been reported, P-BLAST found the amino acid sequences of iron- sulfur subunit (Chain B) and cytochrome b small subunit (Chain D) in Meloidogyne hapla SDH reported in Uniprot (Entry: A0A1I8BD68, A0A1I8C198) had high identity with that in Ascaris suum SDH (Figure 4). Based on Figure 4, it was inferred that SDH between Meloidogyne and Ascaris suum had high homology. In addition, the amino acid residues at the action site of flutolanil in chains B and D of Ascaris suum SDH were almost completely consistent with those in Meloidogyne hapla SDH, such as B/PRO 193, B/TRP 197, B/ILE 242, D/ASP 106, and D/TYR 107. Therefore, Ascaris suum SDH retrieved from the RCSB Protein Data Bank (PDB ID: 3VRB) was adopted as molecular docking receptor.

  Figure 4

  

  Figure 4.  Amino acid sequence comparison of iron-sulfur subunit and cytochrome b small subunit between Meloidogyne hapla SDH and Ascaris suum SDH

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  Subsequently, fluopyram, compounds Ⅰ-1, Ⅱ-1 and Ⅱ-4 were manually docked into the active site in Ascaris suum SDH on the basis of binding positions of flutolanil, and the results were illustrated in Figure 5. It showed that the amide group in compound Ⅰ-1 acted as a hydrogen-bond donor to form hydrogen bond with amino acid residue D/TYR 107, whereas the carbonyl group in fluopyram, and sulfonyl groups in compounds Ⅱ-1, Ⅱ-4 formed hydrogen bonds as acceptors with amino acid residues B/TRP197, D/TYR107, which were consistent with the binding mode of flutolanil in Ascaris suum SDH (PDB ID: 3VRB). In addition, the introduction of other hydrogen bond acceptors or donors on the amide-linked aromatic rings contributed to the interaction between the target molecules and amino acid residues. In combination with biological activity, we speculated that directly attaching the carbonyl or N-sulfonyl groups in the amide bridge to the aromatic rings containing other hydrogen bond acceptors or donors was favorable to the nematocidal activity.

  Figure 5

  

  Figure 5.  Binding mode of fluopyram (A), compounds Ⅰ-1 (B), Ⅱ-1 (C) and Ⅱ-4 (D) with Ascaris suum SDH

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  3.   Conclusions

  In summary, eighteen novel target compounds were designed and synthesized by adopting amide group flipping and introducing N-sulfonyl substituted amide bonds. The bioassays indicated that the structural modification of the amide bridge had different effects on their nematocidal and fungicidal activities. For the fungicidal activity, no compounds displayed excellent and broad-spectrum inhibitory rates. However, the introduction of the N-sulfonyl substituent on the amide group in compounds Ⅱ was favorable to improving the nematocidal activity in comparison with compounds Ⅰ. Combined with our previous work and molecular docking, directly attaching the carbonyl or N-sulfonyl groups in the amide bridge to the aromatic rings was favorable to the nematocidal activity. These results would provide important guidance for the design and exploration of efficient nematocidal lead compounds.

  4.   Experimental section

  4.1   Reagents and instruments

  All reaction reagents were analytical grade. Melting points for target compounds were determined on a X-4 binocular microscope (Gongyi Tech. Instrument Co., Henan, China). ¹H NMR and ¹³C NMR were obtained on a Bruker AV-400 spectrometer (400 MHz), tetramethylsilane as the internal standard. Mass spectra were recorded on a high-resolution mass spectrometer (HRMS) (Varian 7.0T FTMS). Column chromatography purification was carried out using silica gel (200~300 mesh).

  4.2   Synthetic procedures for target compounds

  2-((3-Chloro-5-(trifluoromethyl)pyridin-2-yl)thio)-N-(2-(trifluoromethyl)phenyl)acet-amide (Ⅰ-1) was synthesized as follows.^[16] A mixture of 3-chloro-5-(trifluoromethyl)- pyridine-2-thiol (1.1 mmol), potassium carbonate (1.3 mmol) in N, N-dimethylformamide (DMF, 5 mL) was heated to 80 ℃ and stirred for 10 min, followed by addition of 2-chloro-N-(2-(trifluoromethyl)phenyl)acetamide (1.0 mmol). The reaction was continued to stir for 3 h at 80 ℃. After reaction completion, the solution was poured into ice-water (100 mL) and then extracted with ethyl acetate (60 mL). The organic layer was dried by anhydrous magnesium sulfate, filtered and concentrated to be purified through chromatograph on silica gel using petroleum ether/ethyl acetate (V:V=3:1) as eluent to give white solid (yield 78%). Compounds Ⅰ-2~Ⅰ-12 and Ⅱ-1~Ⅱ-6 were prepared similarly.

  2-((3-Chloro-5-(trifluoromethyl)pyridin-2-yl)thio)-N-(2-(trifluoromethyl)phenyl)acetamide (Ⅰ-1): White granular, yield 78%. m.p. 118~120 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.89 (s, 1H, CONH), 8.63 (s, 1H, pyridine-H), 8.18 (d, J=8.2 Hz, 1H, Ph-H), 7.84 (s, 1H, pyridine-H), 7.55 (dd, J=12.8, 7.9 Hz, 2H, Ph-H), 7.21 (t, J=7.6 Hz, 1H, Ph-H), 4.06 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 166.8, 160.3, 144.0 (q, J=4.0 Hz), 134.9 (q, J=2.0 Hz), 133.2 (q, J=4.0 Hz), 132.8 (q, J=0.8 Hz), 129.4, 126.0 (q, J=6.0 Hz), 124.6, 124.5, 124.2 (q, J=33.0 Hz), 123.8 (q, J=272.0 Hz), 122.7 (q, J=270.0 Hz), 120.2 (q, J=30.0 Hz), 34.7; HRMS calcd for C₁₅H₁₀ClF₆N₂OS [M+H]⁺ 415.0101, found 415.0099.

  2-((4-(Trifluoromethyl)pyrimidin-2-yl)thio)-N-(2- (trifluoromethyl)phenyl)acetamide (Ⅰ-2): White granular, yield 70%. m.p. 123~125 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.83 (d, J=5.0 Hz, 1H, pyrimidine-H), 8.77 (s, 1H, CONH), 8.14 (d, J=8.1 Hz, 1H, Ph-H), 7.55 (t, J=8.4 Hz, 2H, Ph-H), 7.38 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.23 (t, J=7.7 Hz, 1H, Ph-H), 4.03 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 171.8, 166.5, 159.9, 156.3 (q, J=36.0 Hz), 134.8, 132.8, 126.0 (q, J=5.0 Hz), 124.9, 124.8, 123.8 (q, J=272.0 Hz), 119.9 (q, J=274.0 Hz), 112.8 (q, J=3.0 Hz), 110.0, 35.5; HRMS calcd for C₁₄H₁₀F₆N₃OS [M+H]⁺ 382.0443, found 382.0440.

  2-((5-Methoxy-3H-imidazo[4, 5-b]pyridin-2-yl)thio)-N- (2-(trifluoromethyl)phenyl) acetamide (Ⅰ-3): White granular, yield 75%. m.p. 141~142 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 13.13 and 12.75 (s, 1H, imidazole-H), 10.07 (s, 1H, CONH), 7.76~7.61 (m, 4H, Ph-H), 7.41 (t, J=7.1 Hz, 1H, pyridine-H), 6.60 (d, J=8.6 Hz, 1H, pyridine-H), 4.23 (s, 2H, CH₂), 3.84 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.8, 160.4, 135.5, 133.5, 129.3, 126.9, 126.7 (q, J=5.0 Hz), 123.9 (q, J=271.0 Hz), 105.1, 53.7, 35.5; HRMS calcd for C₁₆H₁₄F₃N₄O₂S [M+H]⁺ 383.0784, found 383.0778.

  2-((4-(Pyridin-4-yl)thiazol-2-yl)thio)-N-(2-(trifluoro- methyl)phenyl)acetamide (Ⅰ-4): White granular, yield 72%. m.p. 140~141 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.88 (s, 1H, CONH), 8.65~8.60 (m, 2H, pyridine-H), 8.11 (d, J=8.3 Hz, 1H, Ph-H), 7.76~7.70 (m, 2H, pyridine-H), 7.67 (s, 1H, thiazole-H), 7.55 (t, J=6.9 Hz, 2H, Ph-H), 7.23 (t, J=7.6 Hz, 1H, Ph-H), 4.20 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 166.3, 163.7, 152.3, 149.6, 149.5, 140.9, 134.6, 132.8, 126.1 (q, J=5.0 Hz), 125.1 (q, J=5.0 Hz), 123.6 (q, J=272.0 Hz), 121.0 (q, J=30.0 Hz), 120.6, 117.2, 37.2; HRMS calcd for C₁₇H₁₃F₃N₃OS₂ [M+H]⁺ 396.0447, found 396.0442.

  Ethyl 2-(2-(2-((3-chloro-5-(trifluoromethyl)pyridin-2- yl)thio)acetamido)thiazol-4-yl)-2-(methoxyimino)acetate (Ⅰ-5): White granular, yield 80%. m.p. 134~135 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 10.23 (s, 1H, CONH), 8.66 (d, J=0.8 Hz, 1H, pyridine-H), 7.85 (d, J=1.4 Hz, 1H, pyridine-H), 7.18 (s, 1H, thiazole-H), 4.40 (q, J=7.1 Hz, 2H, CH₂CH₃), 4.11 (s, 2H, CH₂), 4.02 (s, 3H, OCH₃), 1.36 (t, J=7.1 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 166.4, 162.6, 159.8 (q, J=1.1 Hz), 158.1, 146.1, 144.0 (q, J=4.2 Hz), 141.0, 133.4 (q, J=3.4 Hz), 129.5, 124.5 (q, J=33.8 Hz), 122.6 (q, J=271.0 Hz), 115.2, 63.2, 62.1, 33.7, 14.1; HRMS calcd for C₁₆H₁₅ClF₃N₄O₄S₂ [M+H]⁺483.0170, found 483.0165.

  Ethyl 2-(2-(2-((4-(trifluoromethyl)pyrimidin-2-yl)thio)- acetamido)thiazol-4-yl)-2-(methoxyimino)acetate (Ⅰ-6): Yellow sticky liquid, yield 77%. ¹H NMR (400 MHz, CDCl₃) δ: 10.29 (s, 1H, CONH), 8.84 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.40 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 4.41 (q, J=7.1 Hz, 2H, CH₂CH₃), 4.08 (s, 2H, CH₂), 4.01 (s, 3H, OCH₃), 1.37 (t, J=7.1 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 171.5, 166.3, 162.6, 160.1, 158.1, 156.2 (q, J=37.0 Hz), 146.1, 141.0, 119.9 (q, J=274.0 Hz), 114.9, 113.0 (q, J=3.0 Hz), 63.2, 62.1, 34.6, 14.1; HRMS calcd for C₁₅H₁₅F₃N₅O₄S₂ [M+ H]⁺ 450.0512, found 450.0509.

  Ethyl 2-(2-(2-((5-methoxy-3H-imidazo[4, 5-b]pyridin-2- yl)thio)acetamido)thiazol-4-yl)-2-(methoxyimino)acetate (Ⅰ-7): White granular, yield 82%. m.p. 106~108 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 13.14 and 12.89 (s, 1H, imidazole-H), 12.83~12.71 (m, 1H, CONH), 7.87~7.69 (m, 1H, pyridine-H), 7.58 (s, 1H, thiazole-H), 6.60 (d, J=8.6 Hz, 1H, pyridine-H), 4.34 (dd, J=14.2, 7.1 Hz, 4H, CH₂ and CH₂CH₃), 3.94 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 1.30 (t, J=7.1 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO- d₆) δ: 167.4, 162.5, 160.4, 159.0, 153.7, 151.0, 147.7, 147.2, 146.8, 140.5, 130.7, 129.3, 128.6, 123.1, 122.1, 114.8, 110.0, 105.1, 63.2, 62.2, 53.8, 53.4, 35.3, 35.1, 34.9, 26.3, 14.4; HRMS calcd for C₁₇H₁₉N₆O₅S₂ [M+H]⁺ 451.0853, found 451.0849.

  Ethyl 2-(2-(2-((4-(pyridin-4-yl)thiazol-2-yl)thio)- acetamido)thiazol-4-yl)-2-(methoxy-imino)acetate (Ⅰ-8): White granular, yield 73%. Decomposed at 225 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.92 (s, 1H, CONH), 8.50 (d, J=5.9 Hz, 2H, pyridine-H), 8.34 (s, 1H, thiazole-H), 7.76 (d, J=6.0 Hz, 2H, pyridine-H), 7.56 (s, 1H, thiazole-H), 4.37~4.27 (m, 4H, CH₂ and CH₂CH₃), 3.93 (s, 3H, OCH₃), 1.28 (t, J=7.1 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.4, 164.3, 162.6, 159.1, 151.7, 150.6, 146.9, 140.6, 140.4, 120.5, 119.3, 114.9, 63.3, 62.2, 37.1, 14.4; HRMS calcd for C₁₈H₁₈N₅O₄S₃ [M+H]⁺ 464.0515, found 464.0514.

  2-((3-Chloro-5-(trifluoromethyl)pyridin-2-yl)thio)-N-(3-cyano-1-(2, 6-dichloro-4-(trifluoromethyl)phenyl)-1H- pyrazol-5-yl)acetamide (Ⅰ-9): White granular, yield 74%. m.p. 162~164 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 9.07 (s, 1H, CONH), 8.20 (s, 1H, pyridine-H), 7.82 (s, 1H, pyridine-H), 7.65 (s, 2H, Ph-H), 7.07 (s, 1H, pyrazole-H), 3.91 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 160.8, 143.2 (q, J=4.0 Hz), 138.1, 136.4, 135.0, 134.7 (q, J=35.0 Hz), 133.6 (q, J=3.0 Hz), 129.7, 128.1, 125.9 (q, J=3.0 Hz), 124.6 (q, J=34.0 Hz), 122.3 (q, J=271.0 Hz), 121.6 (q, J=272.2 Hz), 112.9, 103.2, 34.1; HRMS calcd for C₁₉H₉Cl₃F₆N₅OS [M+H]⁺ 573.9492, found 573.9494.

  2-((4-(Trifluoromethyl)pyrimidin-2-yl)thio)-N-(3-cyano-1-(2, 6-dichloro-4-(trifluoromethyl)phenyl)-1H-pyrazol-5- yl)acetamide (Ⅰ-10): White granular, yield 71%. m.p. 160~161 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.72 (s, 1H, CONH), 8.60 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.65 (s, 2H, Ph-H), 7.35 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.04 (s, 1H, pyrazole-H), 3.88 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 172.2, 165.7, 159.7, 156.2 (q, J=37.0 Hz), 138.1, 136.3, 135.0, 134.6 (q, J=34.0 Hz), 127.9, 126.0 (q, J=4.0 Hz), 121.7 (q, J=272.3 Hz), 119.7 (q, J=273.8 Hz), 113.0, 112.9 (q, J=2.6 Hz), 103.4, 34.7; HRMS calcd for C₁₈H₉Cl₂F₆N₆OS [M+H]⁺ 540.9834, found 540.9833.

  2-((5-Methoxy-3H-imidazo[4, 5-b]pyridin-2-yl)thio)-N- (3-cyano-1-(2, 6-dichloro-4-(trifluoromethyl)phenyl)-1H- pyrazol-5-yl) acetamide (Ⅰ-11): White granular, yield 67%. m.p. 131~132 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 13.10 and 12.76 (s, 1H, imidazole-H), 11.03 (s, 1H, CONH), 8.22 (s, 2H, Ph-H), 7.71 (d, J=8.6 Hz, 1H, pyridine-H), 7.25 (s, 1H, pyrazole-H), 6.61 (d, J=8.6 Hz, 1H, pyridine-H), 4.22 (s, 2H, CH₂), 3.86 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 166.6, 160.4, 140.4, 135.9, 135.8 (q, J=1.0 Hz), 133.5 (q, J=33.3 Hz), 127.0, 126.9 (q, J=3.3 Hz), 122.6 (q, J=272.4 Hz), 113.9, 110.0, 105.1, 102.2, 53.7, 35.6; HRMS calcd for C₂₀H₁₃Cl₂F₃N₇O₂S [M+H]⁺ 542.0175, found 5642.0186.

  2-((4-(Pyridin-4-yl)thiazol-2-yl)thio)-N-(3-cyano-1-(2, 6-dichloro-4-(trifluoromethyl) phenyl)-1H-pyrazol-5-yl)acet- amide (Ⅰ-12): White granular, yield 69%. m.p. 187~188 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 9.97 (s, 1H, CONH), 8.63 (d, J=4.7 Hz, 2H, pyridine-H), 7.54 (s, 1H, thiazole-H), 7.44 (d, J=6.0 Hz, 2H, pyridine-H), 7.20 (s, 2H, Ph-H), 7.12 (s, 1H, pyrazole-H), 4.06 (s, 2H, CH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 166.9, 165.9, 151.3, 150.7, 138.9, 138.6, 135.9, 134.8, 133.8 (q, J=34.0 Hz), 128.1, 125.6 (q, J=3.0 Hz), 121.7 (q, J=273.0 Hz), 119.7, 117.1, 113.0, 102.9, 37.1; HRMS calcd for C₂₁H₁₂Cl₂F₃N₆OS₂ [M+H]⁺ 554.9838, found 554.9850.

  Methyl 2-(N-(2-((3-chloro-5-(trifluoromethyl)pyridin-2- yl)thio)acetyl)sulfamoyl) benzoate (Ⅱ-1): White granular, yield 66%. m.p. 149~151 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.50 (s, 1H, NH), 8.41 (s, 1H, pyridine-H), 8.33 (s, 1H, pyridine-H), 8.07 (d, J=7.7 Hz, 1H, Ph-H), 7.78~7.68 (m, 3H, Ph-H), 4.14 (s, 2H, CH₂), 3.91 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.4, 166.8, 161.0, 143.9 (q, J=4.0 Hz), 136.6, 134.2, 133.8 (q, J=3.0 Hz), 132.6, 131.1, 131.1, 129.3, 128.4, 123.4 (q, J=271.0 Hz), 122.6 (q, J=33.0 Hz), 53.6, 34.8; HRMS calcd for C₁₆H₁₂ClF₃N₂NaO₅S₂ [M+Na]⁺ 490.9720, found 490.9719.

  Methyl 2-(N-(2-((4-(trifluoromethyl)pyrimidin-2- yl)thio)acetyl)sulfamoyl)benzoate (Ⅱ-2): White granular, yield 63%. m.p. 141~142 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.48 (s, 1H, NH), 8.84 (d, J=5.0 Hz, 1H, pyrimidine-H), 8.13~8.01 (m, 1H, pyrimidine-H), 7.81~ 7.59 (m, 4H, Ph-H), 4.10 (s, 2H, CH₂), 3.87 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.7, 167.3, 166.7, 161.4, 154.6 (q, J=36.0 Hz), 136.7, 134.2, 132.5, 131.2, 131.0, 129.5, 120.5 (q, J=273.0 Hz), 113.5 (q, J=2.0 Hz), 53.6, 35.4; HRMS calcd for C₁₅H₁₂F₃N₃NaO₅S₂ [M+Na]⁺458.0063, found 458.0061.

  Methyl 2-(N-(2-((4-(pyridin-4-yl)thiazol-2-yl)thio)- acetyl)sulfamoyl)benzoate (Ⅱ-3): White granular, yield 68%. m.p. 209~211 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.63 (d, J=5.7 Hz, 2H, pyridine-H), 8.36 (s, 1H, thiazole-H), 8.06 (d, J=7.3 Hz, 1H, Ph-H), 7.87 (d, J=6.0 Hz, 2H, pyridine-H), 7.74~7.57 (m, 3H, Ph-H), 4.24 (s, 2H, CH₂), 3.88 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.4, 166.8, 164.5, 151.6, 150.1, 141.0, 137.0, 134.0, 132.5, 131.1, 130.8, 129.4, 120.8, 119.6, 53.6, 37.9; HRMS calcd for C₁₈H₁₆N₃O₅S₃ [M+H]⁺ 450.0247, found 450.0246.

  N-((4-Acetamidophenyl)sulfonyl)-2-((3-chloro-5- (trifluoromethyl)pyridin-2-yl)thio)acetamide (Ⅱ-4): White granular, yield 70%. Decomposed at 241 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.37 (s, 1H, CONHSO₂), 10.36 (s, 1H, CONH), 8.30 (d, J=1.4 Hz, 1H, pyridine-H), 8.20 (s, 1H, pyridine-H), 7.87 (d, J=8.9 Hz, 2H, Ph-H), 7.78 (d, J=8.9 Hz, 2H, Ph-H), 4.05 (s, 2H, CH₂), 2.09 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.4, 166.5, 161.1, 144.4, 143.7 (q, J=5.0 Hz), 133.7 (q, J=4.0 Hz), 132.8, 129.4, 128.3, 123.3 (q, J=271.0 Hz), 122.5 (q, J=33.0 Hz), 118.6, 34.8, 24.5; HRMS calcd for C₁₆H₁₄ClF₃N₃O₄S₂ [M+H]⁺ 468.0061, found 468.0059.

  N-((4-Acetamidophenyl)sulfonyl)-2-((4-(trifluoro- methyl)pyrimidin-2-yl)thio) acetamide (Ⅱ-5): White granular, yield 60%. m.p. 191~193 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.39 (s, 1H, CONHSO₂), 10.38 (s, 1H, CONH), 8.85 (d, J=5.0 Hz, 1H, pyrimidine-H), 7.84 (d, J=8.9 Hz, 2H, Ph-H), 7.75 (d, J=8.9 Hz, 2H, Ph-H), 7.66 (d, J=5.0 Hz, 1H, pyrimidine-H), 4.04 (s, 2H, CH₂), 2.09 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 171.8, 169.5, 166.6, 161.4, 154.5 (q, J=36.0 Hz), 144.3, 132.9, 129.3, 120.5 (q, J=274.0 Hz), 118.8, 113.5, 35.4, 24.6; HRMS calcd for C₁₅H₁₄F₃N₄O₄S₂ [M+H]⁺ 435.0403, found 435.0405.

  N-((4-Acetamidophenyl)sulfonyl)-2-((4-(pyridin-4-yl)- thiazol-2-yl)thio)acetamide (Ⅱ-6): White granular, yield 55%. m.p. 194~195 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.59 (s, 1H, CONH), 8.86 (s, 2H, pyridine-H), 8.72 (s, 1H, thiazole-H), 8.27 (s, 2H, pyridine-H), 7.78 (d, J=8.4 Hz, 2H, Ph-H), 7.66 (d, J=8.5 Hz, 2H, Ph-H), 4.24 (s, 2H, CH₂), 2.08 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 169.6, 166.6, 165.5, 149.7, 146.1, 144.6, 144.4, 132.7, 129.1, 124.2, 122.5, 118.6, 37.6, 24.6; HRMS calcd for C₁₈H₁₇N₄O₄S₃ [M+H]⁺ 449.0406, found 449.0403.

  4.3   Biological activity screening

  The nematocidal activities of target compounds against Meloidogyne incognita were screened and evaluated referring to literatures.^{[17, 18]} Eggs of M. incognita were extracted from infected roots of tomato (Solanum lycopersicum L.), and placed on a mesh nylon filter (openings 30 μm in diameter) to obtain second-stage juveniles (J2) of M. incognita, which were used for bioassays immediately. In addition, the fungicidal inhibition rates of target compounds were investigated as previously reported.^[19] Common agricultural pathogens, including Rhizoctonia solani, Gibberella zeae, Physalospora piricola, Cercospora circumscissa Sacc., Alternaria kikuchiana Tanaka, Botrytis cinerea, Colletotrichum capsici, Phyllosticta melongenae Sawada, were taken as the test objects.

  4.4   Molecular docking

  Literatures reported that the mechanism of action of nematocide fluopyram was to inhibit complex Ⅱ on the mitochondrial respiratory electron transport chain, namely succinate dehydrogenase (SDH) or succinate coenzyme Q reductase (SQR).^[20] The surflex-dock method^[21] was applied to study the binding mode of target compounds Ⅱ with Ascaris suum SDH using the SYBYL 6.9 software package. The receptor and the ligand molecules were prepared by standard procedures.

  Supporting Information   ¹H NMR and ¹³C NMR spectra of target compounds Ⅰ-1~Ⅰ-12 and Ⅱ-1~Ⅱ-6. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

  
  
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• 

  Figure 1  Chemical structures of fosthiazate, avermectin B2a and fluopyram

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  Figure 2  Structures of reported nematocidal amide compounds

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  Figure 3  Design strategy of the target compounds

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  Scheme 1  Synthetic routes of the target compounds

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  Figure 4  Amino acid sequence comparison of iron-sulfur subunit and cytochrome b small subunit between Meloidogyne hapla SDH and Ascaris suum SDH

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  Figure 5  Binding mode of fluopyram (A), compounds Ⅰ-1 (B), Ⅱ-1 (C) and Ⅱ-4 (D) with Ascaris suum SDH

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  Table 1.  Nematocidal activity of the target compounds against M. incognita

  Compound	Mortality/%
  	200 μg/mL	100 μg/mL
  Ⅰ-1	0	0
  Ⅰ-2	0	0
  Ⅰ-3	0	0
  Ⅰ-4	1.9±0.07	0
  Ⅰ-5	0	0
  Ⅰ-6	1.22±0.08	0
  Ⅰ-7	0	0
  Ⅰ-8	0	0
  Ⅰ-9	0	0
  Fluopyram	99.3±1.14	99.4±1.12
  Ⅰ-10	1.66±0.31	1.36±0.11
  Ⅰ-11	4.44±0.81	0
  Ⅰ-12	2.72±0.93	1.71±0.58
  Ⅱ-1	62.7±1.95	57.0±1.34
  Ⅱ-2	66.5±1.75	40.4±1.04
  Ⅱ-3	57.5±1.47	49.7±0.17
  Ⅱ-4	77.5±0.97	57.2±1.43
  Ⅱ-5	50.5±2.09	48.9±2.31
  Ⅱ-6	50.3±0.56	49.6±1.36

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  Table 2.  Fungicidal activity of the target compounds at 50 μg/mL^a

  Compound	Inhibition rate/%
  	GZ	RS	CC	PM	BC	PP	AK	CS
  Fluopyram	55.6±0.09	44.3±0.09	74.9±0.38	67.5±0.03	92.2±0.15	79.3±0.13	100.0	100.0
  Ⅰ-1	8.3±0.14	19.0±0.05	61.1±0.43	31.7±0.05	9.7±0.24	25.9±0.06	21.5±0.06	22.1±0.10
  Ⅰ-2	4.2±0.24	16.5±0.01	6.3±0.17	48.0±0.13	9.7±0.26	15.5±0.05	21.5±0.06	52.3±0.24
  Ⅰ-3	19.4±0.18	20.3±0.26	38.3±0.40	41.5±0.08	9.8±0.13	36.2±0.05	40.6±0.22	57.3±0.10
  Ⅰ-4	1.4±0.21	21.5±0.13	10.9±0.18	25.2±0.10	9.7±0.42	41.4±0.05	26.8±0.15	55.7±0.08
  Ⅰ-5	38.9±0.15	26.6±0.22	47.4±0.25	64.2±0.05	9.7±0.13	12.1±0.10	0	64.9±0.10
  Ⅰ-6	15.3±0.18	21.5±0.09	47.4±0.41	57.7±0.01	9.7±0.13	75.9±0.06	34.2±0.05	67.9±0.10
  Ⅰ-7	0	21.5±0.14	20.0±0.13	33.3±0.19	9.6±0.15	41.4±0.14	21.5±0.06	19.1±0.15
  Ⅰ-8	22.2±0.28	19.0±0.10	13.1±0.08	38.2±0.10	12.8±0.06	46.6±0.12	68.2±0.10	42.0±0.15
  Ⅰ-9	15.3±0.06	17.0±0.14	33.7±0.39	38.2±0.14	9.7±0.22	20.7±0.09	14.3±0.07	34.4±0.17
  Ⅰ-10	27.8±0.13	39.2±0.57	61.1±0.47	46.3±0.02	9.5±0.21	11.2±0.11	13.0±0.10	86.3±0.05
  Ⅰ-11	22.2±0.25	8.9±0.22	58.9±0.61	57.7±0.08	90.7±0.13	45.7±0.15	34.2±0.05	34.4±0.15
  Ⅰ-12	22.2±0.10	10.1±0.24	33.7±0.14	35.0±0.25	75.1±0.06	43.1±0.10	63.9±0.10	84.7±0.06
  Ⅱ-1	13.9±0.31	8.9±0.02	7.4±0.06	57.7±0.30	7.2±0.07	25.9±0.17	25.7±0.13	26.7±0.10
  Ⅱ-2	22.2±0.27	20.3±0.08	40.6±0.31	28.5±0.22	5.1±0.08	19.0±0.09	8.8±0.19	69.5±0.13
  Ⅱ-3	22.2±0.12	11.4±0.10	81.7±0.33	44.7±0.01	25.3±0.06	70.7±0.10	51.2±0.05	52.7±0.06
  Ⅱ-4	23.6±0.45	0	24.6±0.22	25.2±0.10	29.2±0.13	8.6±0.21	8.8±0.17	86.3±0.10
  Ⅱ-5	6.9±0.11	16.5±0.13	13.1±0.13	51.2±0.01	40.1±0.15	29.3±0.17	30.0±0.10	52.7±0.10
  Ⅱ-6	9.7±0.20	13.9±0.17	7.4±0.15	44.7±0.19	26.8±0.08	55.2±0.13	70.3±0.73	53.2±0.15
  a GZ: Gibberella zeae; RS: Rhizoctonia solani; CC: Colletotrichum capsici; PM: Phyllosticta melongenae Sawada; BC: Botrytis cinerea; PP: Physalospora piricola; AK: Alternaria kikuchiana Tanaka; CS: Cercospora circumscissa Sacc.

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