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• Root-knot nematodes (RKNs) are soil worms belonging to the plant-parasitic nematodes that feed on the roots of different crops such as tomato, pepper, watermelons and onions, resulting in an annual crop loss of about 100 billion USD around the world [1, 2]. At present, fosthiazate and abamectin are the most commonly used nematocides in the market. Avermectin is a broad-spectrum, highly effective pesticide. It is widely used in the prevention and control of harmful insects, mites and nematodes in agriculture [3, 4]. Fosthiazate is an organophosphorus nematicide, which has the advantages of non-cross resistance of most organophosphorus compounds and easy to be degraded [5-7]. Although recently some new stars of nematocide industry like fluensulfone and tioxazafen have entered the pesticide market [8-10], such as tioxazafen, a new type of broad-spectrum nematocide, mainly used for seed treatment, has a good control effect. The search for novel chemotypes of nematicides, which are safer, environmentally friendly, and nematode-specific, is still urgent [11].

  Chromones are a group of naturally occurring compounds that are ubiquitous in nature, especially in plants. Both of synthetic and natural origin have been recognized to display valuable antibacterial, antifungal and antirhinoviral activity (Fig. S1 in Supporting information) [12, 13]. More importantly, Lee et al. [14] reported that chromone derivatives are active nematicidal compounds.

  It is well known that pyrazole exhibited diversity biological activities, such as antimicrobial, xanthine oxidase inhibitory activity, herbicidal [15-19]. What is more, Badische Anilin-undSoda-Fabrik (BASF) crop science reported in 2007 that the pyrazole derivative 1-1 can be used as a nematicide (Fig. S2 in Supporting information) [20]. In our group's previous study, Li et al. [21] and Chen et al. [22] reported that pyrazole derivative containing a diphenylacetylene scaffold (1-2) and pyrazole derivative containing a 1, 2, 3-triazole scaffold (1-3) exhibited good inhibition rates against Meloidogyne incognita (Fig. S2). Liu et al. [23, 24] also synthesized some pyrazole derivatives 1-4 and 1-5 (Fig. S2).

  In this study, our design stratege is to construct a novel structure scaffold which was combined chromone and substituted pyrazole by flexible chiain. In 2014, Syngenta and Bayer crop science announced a series of aromatic/heterocyclic carboxamides, which have good nematocidal activity [25, 26] (Fig. S3 in Supporting information). With all this in mind, we introduced amide and alkyl ester flexible chain into chromone structure as linker to investigate the nematicidal bioactivity, and designed two series of novel chromone derivatives (Scheme 1, A and B). The chromone derivatives containing substituted pyrazole were synthesized and their biological activities in vivo against M. incognita were measured.

  Scheme 1

  

  Scheme 1.  The design strategy of title compounds.

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  The general synthetic routes (Schemes S1 and S2 in Supporting information) and experimental data for synthesis of compounds A1–A14 and B1–B4 were listed in Supporting information. The second-stage juveniles (J2) of M. incognita used in all tests were cultured by Huzhou Modern Agricultural Biotechnology Innovation Center, Chinese Academy of Sciences, China. Assay of nematicidal activity can be found in Supporting information.

  The in vivo nematocidal activity of compounds A1–A14 and B1–B4 against M. incognita are listed in Table 1. Fenamiphos and Avermectin were used as controls. As shown in Table 1, the preliminary bioassays indicated that almost all of the target compounds had exceed 90% inhibition rates against M. incognita at 25.0 mg/L.

  Table 1

  

  Table 1.  Nematicidal activities of compounds A1–A14, B1–B4 against J2 of M. incognita in test tubes.

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  Based on the data in Table 1, when we fixed chromone and substituted pyrazole two parts, and focus our attention on the effect of the linker type on the nematicidal bioactivity, it was found that R₅ and R₆ were hydrogen (compound A1, 100%) displayed good inhibitory activities against M. incognita. However, when R₆ was hydrogen and methyl, ethyl and phenyl were introduced into R₅ as flexible chain, the nematicidal activities of compound A2 (95.5%), A3 (90.3%), A4 (91.4%) were weaker than that of A1, which indicated that R₅ and R₆ of flexible chain without substitutes are in benefit for increasing nematocidal activity. When chromone and flexible chain were fixed, the substituents of pyrazole were changed further. Compared with pyrazole substituted by benzene and pyridine, it was found that the bioactivity of compounds A6 (90.3%) was higher than that of compounds A1 (86.6%) at 10.0 mg/L. In order to identify pyridine group is benefit for increasing nematicidal activity, compound A5, A7, A8, and A9 were synthesized, compounds A5 (70.3%), A8 (70.3%) and A9 (80.4%) had higher nemoticidal activities than compounds A3 (65.6%), A2 (65.7%) and A4 (60.0%), respectively. Meanwhile, when R₆ of flexible chain was methyl, the nematicidal activity of compound A7 (75.9%) was also lower than that of A6 (90.3%), which suggested further that no substitute on both R₅ and R₆ of flexible chain are benefit for increasing nematocidal activity once again. Furthermore, the influence of substitutes position of nitrogen atom of pyrazole ring were investigated, which was shown in Scheme 1. When the substitutes were R₁, compound A1–A14, correspondingly, the substitutes were R₂ in compound B1–B4. Analyzing the activity of these compounds, it was found that the nematicidal activities of B1 (50.3%), B2 (60.5%), B3 (50.4%) and B4 (55.0%) decreased compared to that of compounds A5 (70.3%), A6 (90.3%), A7 (75.9%), A8 (70.3%) at 10.0 mg/L. It suggest that the R₁ of pyrazole is important to the nemoticidal activity.

  In some papers, it was reported that CF3 group can increase nematocidal activity [27, 28]. So we tried to introduce CF3 group, and also introduce CN and Br group into pyrazole. It was found that A10 (100%), A11 (100%), A12 (96.7%) and A13 (100%) displayed excellent nematicidal activity against M. incognita at 25.0 mg/L, respectively. Among the tested compounds, A10 and A11 still exhibited 100% inhibition rates at 10.0 mg/L. It demonstrated the introduction of CF₃ group is really benefit for increasing nematocidal activity. Compared with the structure reported by Liu et al. [23, 24], when pyrazole carboxamide kept, the highest inhibition rates of compounds 1–5 (Fig. S2) and 1–4 (Fig. S2) was 59.4% at 10.0 mg/L and 100% at 40.0 mg/L, our compound A11 reached 100% inhibition rates at 10.0 mg/L under the same test condition. It indicated that chromone and ester flexible chain might increase the nemoticidal activities of our compounds.

  As shown in Table 2, to determine the strength of activity of title compounds, the nematicidal evaluation of the compounds in 100% inhibitory activity at 10.0 mg/L, such as A10 and A11, was continued at lower concentrations of 5.0 mg/L and 1.0 mg/L. However, it was found that the inhibitory activities decreased very quickly with the decreasing of the treatment concentration. The nemoticidal bioactivity of compound A10 was 21.4% at the treatment concentration 5.0 mg/L; that of positive control tioxazafen exhibited 92.9%. The reason why the inhibitory rates decreased so quickly still needs to be investigated further. When the treatment concentration decreased to 1.0 mg/L, compound A11 had 14.3% inhibitation rate, meanwhile, tioxazafen exhibited 28.6% inhibition rates at 1.0 mg/L. Although the data is not very good, it still means that compound A11 is valuable to modify further based on tioxazafen.

  Table 2

  

  Table 2.  Nematicidal activities of compounds A10 and A11 against J2 of M. incognita in test tubes.

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  Some literatures has reported that AChE is one of the target of nematicides [29, 30]. Our high active compound was also tried to investigate the mode with AChE, the binding modes between AChE (PDB:1odc) and the active compound A6 were selected as exemplified in the case of representative compound. First of all, we did the docking poses of A6 with the AChE, and then did the compound A10 and A11. Performed on Maestro 10.2 software, the most likely binding conformation was selected based on the glide score and the binding mode, which was shown in Fig. S4 (Supporting information). From the docking result, one hydrogen bond and four π–π stacking interactions were observed between the target and the compound A6. The H atom of amide group in alkyl chain of the compound A6 can form tightly interaction with the OH group of Tyr121 in the hinge domain of the target via a Hbond. Meanwhile, the benzene ring of the compound A6 formed the π–π stacking interaction with the Trp279 and Tyr70 residue. The pyridine ring of the compound A6 formed the π–π stacking interaction with the Phe330 and Trp84 residue. All of these interactions contributed the stability of the complex between AChE and the compound A6. It is different from the mode reported by Liu et al. [23, 24]. Their docking results indicated that compound 1-4 (Fig. S2) interact with amino acid residue Tyr 121, Trp 279 (compounds 1–5(Fig. S2) interact with amino acid residue Tyr 121) of AChE via hydrogen bond. In order to verify the effect of hydrogen of CH₂CH₂ of flexible chain, we selected A5 and A8 to make a docking. The comparison of the docking A5, A6 and A8 (Fig. S4) suggests that, as the hydrogen on the flexible chain was replaced by methyl or ethyl, the number of hydrogen bonds on the flexible chain dispeared, this also explained the experimental result that when the hydrogen of CH₂CH₂ of flexible chain was replaced by methyl and ethyl groups, the nematicidal activity decreased. We also made the molecular docking of A10 and A11 with the AChE (Fig. S5 in Supporting information), both A10 and A11 interact with amino acid residue Tyr121, Trp279, Tyr70, Trp84 and Phe330 of AChE via hydrogen bond and π–π stacking. It is the same as that of compound A6. The docking results also indicated that all the active compounds had similiar binding mode.

  In conclusion, a novel series of chromone derivatives containing substituted pyrazole were designed and synthesized. The nematocidal activity results showed some of them possessed good activity against M. incognita at 10.0 mg/L. The docking results indicated that both compound A10 and A11 interacts with amino acid residue Tyr121, Trp279, Tyr70, Trp84 and Phe330 of AChE via hydrogen bond and π–π stacking. It implied that chromone containing substituted pyrazole was a potential active structure to be worth studying further.

  Acknowledgments

  This work was financial supported by the National Natural Science Foundation of China (No. 21672061), National Key Research Program of China (No. 2017YFD0200505), and Fundamental Research Funds for the Central Universities (No. 222201718004).

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2017.10.011.

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  Scheme 1  The design strategy of title compounds.

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  Table 1.  Nematicidal activities of compounds A1–A14, B1–B4 against J2 of M. incognita in test tubes.

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  Table 2.  Nematicidal activities of compounds A10 and A11 against J2 of M. incognita in test tubes.

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