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• Plant-parasitic nematodes (PPNs) are one of the major threats to agriculture and account for 14% of the global crop yield losses which is nearly 173 billion dollars annually [1, 2]. Root-knot nematode (Meloidogyne spp.) is one of the most destructive groups of PPNs [3]. If the plants were infected by root-knot nematodes, the characteristic such as stunted growth, wilting, leaf discoloration, and deformation of the roots would appear [4]. In addition, root-knot nematodes can interact with other pathogens and develop disease complexes, such as Fusarium wilt, Rhizoctonia solani, and Thielaviopsis basicola, which caused a greater loss of plants [4, 5].

  Synthetic nematicides play a prominent role in nematode management for their high efficiency and low cost [6]. For decades, PPNs have been mainly controlled by organophosphates, carbamates, and soil fumigants [7]. However, most of them were gradually banned or restricted due to their environmental risks and high toxicity [3, 8]. So far, only a few nematicides have developed, including Fosthiazate, Fluensulfone, Fluopyram, Tioxazafen, Cyclobutrifluram, and Fluazaindolizine (Fig. 1) [9-12]. Therefore, the research and development of new nematicidal molecules are essential for the management of PPNs.

  Figure 1

  

  Figure 1.  Chemical structures of commercialized nematicides (A) and molecular design of title compounds (B).

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  At present, the mode of actions of six new commercialized nematicides except Fosthiazate and Fluopyram are still unknown. The absence of target information makes the lead discovery based on target difficult. Thus, bioisosterism and scaffold hopping might be an applicable strategy for the exploration of nematicidal lead [13]. Ring closure is a common scaffold hopping strategy for discovering novel structures in agrochemical design [14]. The physicochemical properties and the binding free energy of the molecule would be changed through ring closure which further affects the activity and stability [15, 16].

  Chalcone is a scaffold with various pesticidal activity including insecticidal, fungicidal, herbicidal, and nematicidal activity [17-20]. However, chalcone is reported to have poor photostability and could be converted to various by-products under UV light [16]. It is unacceptable as a pesticide. To overcome this disadvantage, based on the structure of chalcone, we designed a series of fused ring derivatives containing 2-carbonyl heterocycles via ring closure strategy (Fig. 1). Then further optimization was carried out based on bioassay results. Forty-two compounds containing fused rings were synthesized. In vitro and in vivo nematicidal activity was evaluated and the structure-activity relationship (SAR) was investigated. In addition, in order to explore the possible mode of action, the influence on the egg hatching rate, motility, and feeding behavior of C. elegans was also studied.

  The synthesis route of title compounds was shown in Scheme 1. The synthetic procedures of intermediates and title compounds were presented in the supporting information. Compounds 1–8 and 17–41 were synthesized by Rap-Stoermer reaction [21, 22]. Firstly, intermediates 1a-3a, 5a, 6a, 8a and 34a-41a were synthesized from corresponding 2-acetylaromatic heterocycles and CuBr₂ in ethyl acetate or THF. Meanwhile, intermediate 4a was synthesized from pyrrole and 2-bromoacetyl bromide by Friedel-Crafts acylation with a low yield. Intermediate 7a was purchased. Then, the corresponding intermediates reacted with different substituted salicylaldehyde in acetonitrile and got compounds 1–8 and 17–41. The yields of most of the compounds were over 70% except those of compounds 6, 31 and 32 which were 55%, 45% and 51%, respectively. Coincidentally, a small quantity of 12a, a key intermediate of compound 12, was detected by GS-MS during the synthesis of intermediate 3a. We optimized the synthetic procedure of intermediate 3a and applied it to the synthesis of intermediate 12a. When 2.4 equiv. of CuBr₂ and 1 equiv. of acetic acid were added into brominate in acetonitrile, the yield of intermediate 12a raised to 65%.

  Scheme 1

  

  Scheme 1.  Synthesis of the target compounds 1–42. Reagents and conditions: (a) corresponding salicylaldehyde, MeCN, 81 ℃; (b) CDMT, NMM, tolune, 1 h; (c) corresponding boric acid, Pd(PPh₃)Cl₂, K₃PO₄, tolune, 110 ℃; (d) CuBr₂, AcOH, MeCN, 81 ℃; (e) 2-aminophenol, diethylamine, DMF, 90 ℃; (f) benzothiazole, I₂, KOH, DMSO, H₂O, 100 ℃; (g) o-phenylenediamine, HCl, H₂O, reflux; (h) Cs₂CO₃, DMF, 90 ℃; (i) 2-thiophenesulfonyl chloride, [(phen)Cu^IBr], K₂CO₃, CH₂Cl₂, H₂O, r.t.; (j) TsNHNH₂, MeOH, 60 ℃, (k) 2-ethynylthiophene, CuBr, Cs₂CO₃, MeCN, 81 ℃.

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  Compounds 9–11 and 42 were synthesized by specific Suzuki coupling [23]. The corresponding acid reacted with 2‑chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT) and then reacted with 2-thiopheneboronic acid or benzofuran-2-boronic acid to obtain product in one pot. The yield of compounds 9–11 was more than 50%. However, that of compound 42 was just 35%. This might be due to the electron-withdrawing effect of the nitro group on the thiophene ring. Compound 12 was obtained by the cyclization of 2-aminophenol with intermediated 12a which was prepared by the above method and the yield of compound 12 was 42% [24]. Compound 13 was synthesized from benzothiazole and 2-acetylthiophene in a one-step reaction with a yield of 65% [25]. Compound 14 was oxidized through intermediate 14a which was obtained by the synthetic method of dibazol with a yield of 35% [26, 27]. Compound 15 was prepared from benzofuran-2-boronic acid and 2-thiophenesulfonyl chloride with a copper catalyst and the yield of compound 15 was 22% [28]. Compound 16 was prepared in two steps. The reaction of salicylaldehyde with p-toluenesulfonyl hydrazide gave the corresponding hydrazone, intermediate 16a. Then it was cyclized with 2-ethynylthiophene to achieve compound 16. The yield of compound 16 was 55% [29].

  The in vitro nematicidal activity of compounds 1–16 against M. incognita was shown in Table 1. The bioassay method of nematicidal activity was introduced in supporting information. To explore the SAR, different aromatic rings, fused rings, and linkers were introduced into the structure and sixteen compounds were synthesized and tested. Compounds 1–8 were firstly synthesized to explore the influence of different aromatic rings on nematicidal activity. Among these compounds, only compounds 2 and 3, in which aromatic rings were thiophene and furan, showed in vitro nematicidal activity against M. incognita after 72 h exposure, and the LC₅₀/72 h (LC₅₀ value at 72 h) of compounds 2 and 3 were 19.46 and 13.42 mg/L, respectively. The nematicidal activity of compound 3 bearing thiophene was superior to that of compound 2 bearing furan. Meanwhile, the influence of substitution positions of aromatic rings was confirmed by the obvious activity difference between compounds 3 and 8, that is, the nematicidal activity of 2-substituted analogues was better than that of 3-substituted analogues. Then SAR of different fused ring replacements was explored based on the bioactivity results of compounds 3 and 9–14. Compound 3 with benzofuran, compound 9 with benzothiophene, and compound 13 with benzothiazole exhibited better in vitro nematicidal activity against M. incognita after 72 h exposure and the mortality was 85.68%, 82.73%, and 70.38% at 40 mg/L, respectively. The LC_{50/72 h} of compounds 3, 9 and 13 were 13.42 mg/L, 18.04 mg/L, and 27.47 mg/L, respectively. Compound 3 possessed the best nematicidal activity among these three compounds. Finally, the linker was optimized by replacing the carbonyl group with the sulfonyl group (compound 15) or the methylene group (compound 16). But neither of these two compounds exhibited in vitro nematicidal activity against M. incognita after 72 h exposure. This suggests that the carbonyl group was important for the nematicidal activity. Based on the above results, compound 3, whose structure was benzofuran-2-yl(thiophen-2-yl)methanone, was confirmed as the most active scaffold among the synthesized compounds and then the derivatives of compound 3 (compounds 17–42) were synthesized and their nematicidal activity was tested subsequently.

  Table 1

  

  Table 1.  In vitro nematicidal activity of compounds 1−16 against M. incognita after 72 h.

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  The in vitro nematicidal activity of compounds 3 and 17–42 against M. incognita was shown in Table 2. When the substituents were introduced into benzofuran ring of compound 3, compounds 18 (R¹ = 5-Cl), 19 (R¹ = 6-Cl), 21 (R¹ = 5-Br), 22 (R¹ = 6-Br), 23 (R¹ = 5-F), 24 (R¹ = 6-F), 26 (R¹ = 5-CH₃), 27 (R¹ = 6-CH₃) and 30 (R¹ = 5-OCF₃) exhibited good nematicidal activity against M. incognita after 72 h exposure. It was obvious that 5- and 6-substituted compounds demonstrated better nematicidal activity than others after 72 h exposure. Furthermore, the nematicidal activity difference at these two substitution positions was confirmed. The in vitro nematicidal activity of 5-substituted compounds was better than those of 6-substituted compounds. Compound 26 (5-CH₃, 96.00%) was superior to that of compound 27 (6-CH₃, 59.03%). Among 5- and 6-substituted compounds, the mortality of compounds with halogen substituents reached 100.00% after 72 h exposure. Moreover, both compounds with weak electron-withdrawing or electron-donating substituents [(compounds 26 (R¹ = 5-CH₃), 27 (R¹ = 6-CH₃), and 30 (R¹ = 5-OCF₃)] possessed good nematicidal activity against M. incognita at 72 h, the mortality of them were 96.00%, 59.03% and 62.41% at 40 mg/L, respectively. But those of compounds with strong electron-withdrawing or electron-donating substituents [(compounds 31 (R¹ = 5-NO₂), 32 (R¹ = 5-CN), 33 (R¹ = 5-CF₃) and 29 (R¹ = 5–OCH₃)] lost their activity at 72 h. In order to further compare the activity of these active compounds, LC_{50/72 h} of these active compounds were tested. Among these nine compounds, the activity of compounds 18, 23 and 24 was better than others and the LC_{50/72 h} values were 4.60, 3.20 and 3.80 mg/L, respectively, which was similar to that of Fosthiazate. It was also found when the substituents of the compounds located at position 5, the nematicidal order could be briefly summarized as follows: -F > -Cl > -Br > -H > -CH₃ > -OCF₃ > > others. It indicated that only the halogen substituents on the benzofuran ring would enhance the nematicidal activity compared with compound 3. Unexpectedly, the substituents on thiophene decreased in vitro nematicidal activity of compound 3. Among compounds 34–42, none exhibited in vitro nematicidal activity after 72 h exposure. Compound 23 (R¹ = 5-F) exhibited the best in vitro nematicidal among all forty-two compounds.

  Table 2

  

  Table 2.  In vitro and in vivo nematicidal activity of compounds 17–42 against M. incognita after 72 h.

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  The in vivo nematicidal activity of compounds 3, 18, 19, 21, 22, 23, 24 and 26 were subsequently tested and presented in Table 2. Most of these compounds exhibited obvious control effects against M. incognita at 40 mg/L in the matrix. The inhibition rates of compounds 18, 19, 21, 23 and 24 were greater than 65%, which were 73.42%, 68.25%, 69.80%, 100.00%, 88.22%, respectively. The SAR rule of the in vivo nematicidal activity was the same as that of in vitro, the activity of 5-substituted compounds was superior to that of 6-substituted. For example, the inhibition rate of compound 23 (R¹ = 5-F, 100.00%) was higher than that of compound 24 (R¹ = 6-F, 88.22%). At the same time, the bioactivity of compound 23 bearing fluoro group was better than compounds bearing other groups at the same substitution position.

  Generally, it is hard to directly observe the behavior of M. incognita in lab conditions because they spend most of their time in soil and roots [30]. C. elegans is a typically model organism in the research of PPNs and nematicide [10, 31]. In order to explore the influence of title compounds on the nematode, the effect of compound 23 with the best nematicidal activity on egg hatching, motility, and feeding behavior was investigated, which might be relative to the process of infecting the root of plants. The detail of the test was described in the supporting information. Fluensulfone was selected as a positive control, which has been reported to inhibit egg hatching, motility, and feeding behavior of C. elegans [32].

  The results in Table S1 (Supporting information) suggested that compound 23 inhibited significantly egg hatching of C. elegans after 24 h and 48 h exposure. The egg hatching rate did not increase with the extension of time. When the concentration of compound 23 was 10 mg/L, only 5.83% of eggs were hatched at 48 h. Meanwhile, egg hatching rates treated with Fluensulfone at the same concentration and pure M9 buffer (with DMSO, negative control) were 87.82% and 91.52% at 48 h, respectively. Evidently, eggs treated with compound 23 have a lower egg hatching rate than that of Fluensulfone. When the concentration of compound 23 was higher than 2.5 mg/L, the inhibition rate of egg hatching was more than 70%. The egg hatching inhibition was still observed even when the concentration was reduced to 0.625 mg/L. It is distinct that the higher concentration of compound 23 increased, the lower the egg hatching rate was. We guessed that compound 23 may act on nematode chitinase due to its high egg hatching inhibition rate.

  In order to further evaluate the influence of compound 23 on the nematodes, we also counted thrashes and pumping frequency of C. elegans to investigate its effect on motility and feeding behavior. As shown in Table S2 (Supporting information), the thrashes of C. elegans decreased with the increase of the concentrations of compound 23 and Fluensulfone. The thrashes were obviously inhibited when the concentration of compound 23 was 40 mg/L, which was nearly 42 thrashes during 30 s with an approximately 34% of inhibition rate. Meanwhile, Fluensuflone has a similar inhibition effect on thrashes, which was nearly 39 thrashes during 30 s. Furthermore, the effect of compound 23 on the feeding behavior of C. elegans was also investigated. However, compound 23 slightly inhibited the pumping frequency of C. elegans, the effect was very weak compared with that of Fluensuflone, which was shown in Table S3 (Supporting information).

  Although some explorations have been performed, the mode of action of compound 23 is unknown. Based on the activity in vitro, it suggested that compound 23 could act directly on nematodes themselves, but the interaction with target enzyme is weak compared to that of Fosthiazate. In addition, due to the high egg hatching inhibition rate of compound 23, we guessed that nematode chitinase may be a possible target of our compounds. There are more works to do to confirm our speculation.

  In conclusion, a series of novel nematicidal compounds with benzofuran-2-yl(thiophen-2-yl)methanone derivatives were rationally designed. Some of the synthesized compounds displayed excellent nematicidal activity which was similar to the commercialized nematicide Fosthiazate. Among these compounds, compound 23 showed the best activity against M. incognita both in vitro and in vivo, meanwhile, it also exhibited inhibiting influence on egg hatching, and motility of C. elegans which implied that compound 23 might have a similar influence on M. incognita and further inhibited the behavior of M. incognita in the process of infecting the root of plants. These results suggested that the structure of benzofuran containing 2-carbonyl thiophene could be considered as a potential nematicidal lead for further structural optimization.

  Declaration of competing interest

  The authors declare that they have no competing interests.

  Acknowledgments

  This work was financial supported by the National Natural Science Foundation of China (No. 21672061). National Key Research Program of China (No. 2018YFD0200105). This work was also supported by Innovation Program of Shanghai Municipal Education Commission (No. 201701070002E00037) and the Fundamental Research Funds for the Central Universities.

  We are very thankful to Dr. Bingli Gao and his co-workers in Huzhou Modern Agricultural Biotechnology Innovation Center, Chinese Academy of Sciences, China for the discussion of nematicidal evaluation and Mengdan Li and Zhifan Mao in East China University of Science and Technology for the help on C. elegans experiments.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.107800.

  
  
• 1. [1]

     C.M. Mesa-Valle, J.A. Garrido-Cardenas, J. Cebrian-Carmona, M. Talavera, F. Manzano-Agugliaro, Agronomy 10 (2020) 1148. doi: 10.3390/agronomy10081148

  2. [2]

     V.T. Nguyen, N.H. Yu, Y. Lee, et al., Front. Microbiol. 12 (2021) 726504. doi: 10.3389/fmicb.2021.726504

  3. [3]

     B. Torto, L. Cortada, L.K. Murungi, S. Haukeland, D.L. Coyne, J. Agric. Food Chem. 66 (2018) 8672–8678. doi: 10.1021/acs.jafc.8b01940

  4. [4]

     J.T. Jones, A. Haegeman, E.G.J. Danchin, et al., Mol. Plant Pathol. 14 (2013) 946–961. doi: 10.1111/mpp.12057

  5. [5]

     T. Liu, H. Wu, H. Jiang, et al., J. Agric. Food Chem. 67 (2019) 6160–6168. doi: 10.1021/acs.jafc.9b01306

  6. [6]

     G. Wang, X. Chen, Y. Deng, Z. Li, X. Xu, J. Agric. Food Chem. 63 (2015) 6883–6889. doi: 10.1021/acs.jafc.5b01762

  7. [7]

     Y. Oka, Y. Saroya, Pest Manage. Sci. 75 (2019) 2095–2106.

  8. [8]

     E. Feist, J. Kearn, Y. Gaihre, V. O'Connor, L. Holden-Dye, Pestic. Biochem. Physiol. 165 (2020) 104541. doi: 10.1016/j.pestbp.2020.02.007

  9. [9]

     A.C. O'Sullivan, O. Loiseleur, R. Staiger, T. Luksch, T. Pitterna, Patent, WO2013143811A1, 2013.

  10. [10]

      U. Slomczynska, M.S. South, G.J. Bunkers, et al., ACS Symp. Ser. 1204 (2015) 129–147. doi: 10.1021/bk-2015-1204.ch010

  11. [11]

      Y. Oka, Agronomy 10 (2020) 1387. doi: 10.3390/agronomy10091387

  12. [12]

      G.P. Lahm, J. Desaeger, B.K. Smith, et al., Bioorg. Med. Chem. Lett. 27 (2017) 1572–1575. doi: 10.1016/j.bmcl.2017.02.029

  13. [13]

      X. Cao, H. Yang, C. Liu, et al., J. Agric. Food Chem. 70 (2022) 11042–11055. doi: 10.1021/acs.jafc.2c00785

  14. [14]

      C. Lamberth, Pest Manage. Sci. 74 (2018) 282–292. doi: 10.1002/ps.4755

  15. [15]

      H. Sun, G. Tawa, A. Wallqvist, Drug Discov. Today 17 (2012) 310–324. doi: 10.1016/j.drudis.2011.10.024

  16. [16]

      P. Huo, Q. Hu, S. Shu, et al., Bioorg. Med. Chem. 29 (2021) 115853. doi: 10.1016/j.bmc.2020.115853

  17. [17]

      Q. Zhou, X. Tang, S. Chen, et al., J. Agric. Food Chem. 70 (2022) 1029–1036. doi: 10.1021/acs.jafc.1c05933

  18. [18]

      P. Caboni, N. Aissani, M. Demurtas, N. Ntalli, V. Onnis, Pest Manage. Sci. 72 (2016) 125–130. doi: 10.1002/ps.3978

  19. [19]

      Z. Wang, R. Yang, P. Li, et al., Pest Manage. Sci. 76 (2020) 1513–1522. doi: 10.1002/ps.5668

  20. [20]

      G.T.T. Nguyen, G. Erlenkamp, O. Jack, et al., Sci. Rep. 6 (2016) 27333. doi: 10.1038/srep27333

  21. [21]

      S. Wei, X. Feng, H. Du, Org. Biomol. Chem. 14 (2016) 8026–8029. doi: 10.1039/C6OB01556E

  22. [22]

      A. Carrer, D. Brinet, J.C. Forent, P. Rousselle, E. Bertounesque, J. Org. Chem. 77 (2012) 1316–1327. doi: 10.1021/jo202060k

  23. [23]

      H. Wu, B. Xu, Y. Li, et al., J. Org. Chem. 81 (2016) 2987–2992. doi: 10.1021/acs.joc.5b02667

  24. [24]

      J. Jiang, H. Zou, Q. Dong, et al., J. Org. Chem. 81 (2016) 51–56. doi: 10.1021/acs.joc.5b02093

  25. [25]

      Q. Gao, X. Wu, F. Jia, et al., J. Org. Chem. 78 (2013) 2792–2797. doi: 10.1021/jo302754c

  26. [26]

      D.P. Hruszkewycz, K.C. Miles, O.R. Thiel, S.S. Stahl, Chem. Sci. 8 (2017) 1282–1287. doi: 10.1039/C6SC03831J

  27. [27]

      A.D. Santos, L.E. Kaim, L. Grimaud, Org. Biomol. Chem. 11 (2013) 3282–3287. doi: 10.1039/c3ob27404g

  28. [28]

      F. Hu, X. Lei, ChemCatChem 7 (2015) 1539–1542. doi: 10.1002/cctc.201500174

  29. [29]

      L. Zhou, Y. Shi, Q. Xiao, et al., Org. Lett. 13 (2011) 968–971. doi: 10.1021/ol103009n

  30. [30]

      W. Cheng, X. Yang, L. Zeng, et al., Antibiotics 9 (2020) 605. doi: 10.3390/antibiotics9090605

  31. [31]

      G. Salinas, G. Risi, Parasitology 145 (2018) 979–987. doi: 10.1017/s0031182017002165

  32. [32]

      J. Kearn, E. Ludlow, J. Dillon, V. O'Connor, L. Holden-Dye, Pestic. Biochem. Physiol. 109 (2014) 44–57. doi: 10.1016/j.pestbp.2014.01.004

• 

  Figure 1  Chemical structures of commercialized nematicides (A) and molecular design of title compounds (B).

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  Scheme 1  Synthesis of the target compounds 1–42. Reagents and conditions: (a) corresponding salicylaldehyde, MeCN, 81 ℃; (b) CDMT, NMM, tolune, 1 h; (c) corresponding boric acid, Pd(PPh₃)Cl₂, K₃PO₄, tolune, 110 ℃; (d) CuBr₂, AcOH, MeCN, 81 ℃; (e) 2-aminophenol, diethylamine, DMF, 90 ℃; (f) benzothiazole, I₂, KOH, DMSO, H₂O, 100 ℃; (g) o-phenylenediamine, HCl, H₂O, reflux; (h) Cs₂CO₃, DMF, 90 ℃; (i) 2-thiophenesulfonyl chloride, [(phen)Cu^IBr], K₂CO₃, CH₂Cl₂, H₂O, r.t.; (j) TsNHNH₂, MeOH, 60 ℃, (k) 2-ethynylthiophene, CuBr, Cs₂CO₃, MeCN, 81 ℃.

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  Table 1.  In vitro nematicidal activity of compounds 1−16 against M. incognita after 72 h.

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  Table 2.  In vitro and in vivo nematicidal activity of compounds 17–42 against M. incognita after 72 h.

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