English

• Grains, vegetables and fruits are important food sources that are tremendously threatened by the infection of plant pathogenic fungi [1]. These pathogenic fungi, such as Rhizoctonia solani (Rs), Fusarium graminearum (Fg), Botrytis cinerea (Bc) and Colletotrichum capsici (Cc), not only lead annually to agricultural yield reductions of at least 10% [2], but also pose huge risks to food security and human health by generating hazardous mycotoxins in infected crops [3]. Nowadays, the rational application of commercialized fungicides is regarded as the most effective approach to alleviate agricultural disease outbreaks caused by phytopathogenic fungi [4, 5]. For example, carbendazim, penthiopyrad and azoxystrobin were developed as the highly-efficient and broad-spectrum fungicides that were widely used to reduce the losses of agricultural economies in last decades. However, the long-term application of existing agricultural fungicides not only leads to the rapid deterioration of fungal resistance [6], but also generates the harmful influences on the environment and non-target organisms [7, 8]. Therefore, it remains a challenge in agricultural sciences to develop highly-efficient and broad-spectrum fungicides with a novel molecular structure [9, 10].

  Featuring high lipid solubilities and desirable enzymatic hydrolyzation within living organisms [11, 12], 1, 3, 5-thiadiazine-2-thione derivatives were documented to possess various bioactivities including anti-proliferative [13], antibacterial [14], antiepileptic [15], antifungal [16], antileishmanial [17], antimalarial [18], antitubercular [19], trypanocidal [20], antioxidant [21] and herbicidal [22] properties. Recent practical studies on 1, 3, 5-thiadiazine-2-thione derivatives showed that this type of nonaromaic heterocycles exhibited desirable inhibition effects against phytopathogenic fungi, bacteria and nematodes. As the important bioactive molecules bearing a 1, 3, 5-thiadiazine-2-thione fragment, milneb and dazoment (Fig. 1) were respectively developed as agricultural fungicide and acaricide over the last three decades [23, 24]. Meanwhile, Mao et al. conducted the antibacterial evaluation of dazoment against Ralstonia solanacearum in field trials and found dazoment could be used as an alternative bactericide to control ginger bacterial wilt [25]. Subsequently, Hwang et al. also found that dazoment could effectively manage agricultural diseases caused by the soil-borne fungal pathogens Plasmodiophora brassicae, Fusarium avenaceum, Pythium ultimum and Rhizoctonia silani [26]. However, to the best of our knowledge, 1, 3, 5-thiadiazine-2-thione derivatives except milneb and dazoment were rarely reported on their structural modifications and agricultural bioactivities.

  Figure 1

  

  Figure 1.  Design strategy of title compounds.

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  Hydrazide derivatives are significant nitrogenous compounds that exhibit herbicidal [27], anticancer [28], antimalarial [29], anti-inflammatory [30], antiviral [31], antibacterial [32], antifungal [33], anticoagulant [34] and insecticidal [35] bioactivities. Among these bioactive hydrazide derivatives, arylhydrazide derivatives have recently attracted tremendous interests from biologists and chemists due to their excellent inhibition effects against various phytopathogenic fungi. For example, ethyl 2-phenylhydrazine-1-carboxylate named Miexiuyihao (Fig. 1) was developed as the systemic fungicide that was mainly applied in effectively controlling wheat stripe rust [36]. Recently, Wang et al. reported that arylhydrazide derivatives bearing a 1, 2, 3-triazole moiety exhibited fine systemic conductions and effectively inhibited the energy generation of various plant fungi [37]. In addition, our previous studies also found that 2-(4-oxoquinazolin-3(4H)-yl)-N'-phenylacetohydrazide derivatives exhibited the impressive anti-phytopathogenic effects against Rs and Fg in vitro [38].

  Based on the above-mentioned outstanding fungicidal activities and excellent properties of 1, 3, 5-thiadiazine-2-thioneandhydrazide derivatives, the aims of this work are to: (ⅰ) construct novel bioactive molecules by combinating the 1, 3, 5-thiadiazine-2-thione fragment with the hydrazide substructure, as shown in Fig. 1, which could enhance the molecular lipid solubility within organisms and improve the combining capacitywith receptorproteins; (ⅱ) generate a series of novel 1, 3, 5-thiadiazine-2-thione derivatives bearing a hydrazide moiety (Scheme 1) and evaluate their antifungal activities in vitro against Rs, Fg, Bc and Cc; and (ⅲ) investigate their in vivo anti-Rs effects and perform their structure-activity relationship (SAR) analysis against the above tested fungi.

  Scheme 1

  

  Scheme 1.  Synthesis route of title compounds.

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  As shown in Scheme 1, the nucleophilic reaction of the primary amide 1 (aniline, para-fluoroaniline or benzylamine) with carbon disulfide in sodium hydroxide solution generated the substituted potassium carbamodithioate 2 that directly reacted with formaldehyde to synthesize the substituted N-hydroxymethyl-S-hydroxymethyl carbamodithioate 3. The substituted 2-(6-thioxo-1, 3, 5-thiadiazinan-3-yl)acetic acid 4 was conveniently synthesized by the nucleophilic substitution of an intermediate 3 with glycine in phosphate buffer (pH 7.8). Using O-(benzotriazol-1-yl)-N, N, N', N'-tetramethyluroniumtetrafluoroborate (TBTU) as the catalyst and triethylamine as the acid binding agent, the intermediates 4 reacted with substituted phenylhydrazines to obtain the target compounds 5–7 with good yields ranging from 31% to 88%. The obtained 1, 3, 5-thiadiazine-2-thione derivatives bearing a hydrazide moiety were confirmed via corresponding FT-IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR and HRMS. The relevant spectroscopic data of synthesized compounds 5–7 were collected and presented in Supporting information.

  Using the agricultural fungicides carbendazim, penthiopyrad and azoxystrobin as positive controls, the in vitro antifungal effects of title compounds against Rs, Fg, Bc and Cc were evaluated by a mycelium growth rate method [5, 10, 37-43]. As shown in Table 1, the title compounds 5b, 6a–6c and 7a–7c exhibited impressive anti-Fg effects in vitro, with the corresponding EC50 values of 2.11, 2.34, 2.17, 2.82, 1.49, 1.19 and 1.73 μg/mL. Meanwhile, the title compounds 5a–5e, 5g, 6a–6c, 6g and 7a–7c obviously inhibited Bc in vitro with the EC₅₀ values of 1.38, 0.85, 0.66, 2.57, 2.26, 2.08, 2.03, 1.43, 2.13, 2.62, 1.75, 1.10 and 1.44 μg/mL, respectively. In addition, Table 1 also showed that the title compounds 5d, 5g, 5h, 5j, 6d, 6g and 7d had obvious anti-Cc effects, with the corresponding EC₅₀ values of 2.29, 2.88, 0.70, 2.12, 0.70, 1.59 and 1.41 μg/mL. Strikingly, the anti-Rs EC50 values of title compounds 5b, 5c, 6d, 7b, 7c and 7d reached 0.24, 0.44, 0.25, 0.28, 0.35 and 0.46 μg/mL, respectively, which are better than that of carbendazim (0.55 μg/mL).

  Table 1 showed that most of title compounds displayed good selectivity and specificity in vitro aganist Rs relative to Fg, Bc and Cc. Impressively, the title compound 5b obviously inhibited the Rs growth in vitro with the EC₅₀ value of 0.24μg/mL, which is approximately 2-folds more effective than the commercialized fungicide carbendazim (0.55 μg/mL). Aiming to further investigate the application values of title compounds as agricultural fungicides, the in vivo anti-Rs effects of title compound 5b, carbendazim and hymexazol were evaluated on rice leaves by a detached leaf assay [44]. As shown in Table 2 and Fig. 2, the in vivo anti-Rs control efficacy of title compound 5b at 100 μg/mL was 61.27%, which is inferior to that of carbendazim at 100 μg/mL (98.08%). Meanwhile, the in vivo bioassay results in Table 2 also presented that the title compound 5b obviously inhibited the Rs growth on rice leaves at 200 μg/mL with an impressive control efficacy of 98.58%, which is obviously better than hymexazol at the same concentration of 200 μg/mL (54.90%). The in vivo bioassay research provides a significant reference for the practical application of 1, 3, 5-thiadiazine-2-thione derivatives bearing a hydrazide moiety in agricultures.

  Table 1

  

  Table 1.  In vitro antifungal EC₅₀ values (μg/mL) of title compounds 5–7.^a

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  Table 2

  

  Table 2.  In vivo anti-Rs control efficacies of bioactive compounds on rice leaves.^a

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  Figure 2

  

  Figure 2.  In vivo anti-Rs effect photographs of bioactive compounds on rice leaves. (A) Blank control; (B) 5b at 100 μg/mL; (C) 5b at 200 μg/mL; (D) Hymexazol at 200 μg/mL; (E) Carbendazim at 100 μg/mL.

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  As shown in Table 1, introducing different substituents at R¹ and R² positions greatly influenced the inhibition effects of title compounds against phytopathogenic fungi. Based on the bioassay results in Table 1, some structure-activity relationships were analyzed and presented as below. First, all title compounds except 5e, 5f and 5h exhibited better inhibition effects against Rs than that against Fg, Bc and Cc. Second, most of title compounds bearing a Bn group at R¹ position, such as 6a, 6d, 6e, 6f and 6g, displayed better anti-Rs and anti-Cc effects than those bearing a Ph (5a, 5d, 5e, 5f and 5g) or 4-FPh (7a and 7d) fragment at R¹ position. Third, the bioassay results in Table 1 showed that a presence of 4-FPh group at R1 position overall enhanced the anti-Fg effects of title compounds. For example, the anti-Fg EC₅₀ values of title compounds 7a, 7b and 7c (R¹ = 4-FPh) respectively reached 1.49, 1.19 and 1.73 μg/mL, which are better than that of title compounds 5a, 5b and 5c (R¹ = Ph; 3.16, 2.11 and 3.66 μg/mL) as well as 6a, 6b and 6c (R¹ = Bn; 2.34, 2.17 and 2.82 μg/mL). Forth, when R¹ was substituted with a Ph group, the anti-Bc EC50 values of obtained compounds 5a–5d were 1.38, 0.85, 0.66 and 2.57 μg/mL, respectively, which are better than that of title compounds 6a–6d (R¹ = Bn; 2.03, 1.43, 2.13 and 4.12 μg/mL) and 7a–7d (R¹ = 4-FPh; 1.75, 1.10, 1.44 and 3.78 μg/mL). Fifth, introducing a 4-F, 4-Cl or 4-Br group at R² position greatly improved the antifungal effects of title compounds against Rs, Fg and Bc. Table 1 showed that the title compounds containing a 4-F (5a, 6a and 7a), 4-Cl (5b, 6b and 7b) or 4-Br (5c, 6c and 7c) group at R² position exhibited better inhibition effects against Rs, Fg and Bc than those molecules bearing a 4-CF3 (5d, 6d and 7d), 4-Me (5e and 6e), 4-OMe (5f and 6f), 2, 4-di-Cl (5g and 6g), 2, 4, 6-tri-Cl (5h), H (5i), 2-F (5j) or 2-Cl (5k) group at R² position.

  In conclusion, aimingto search for novel bioactive molecules with anti-phytopathogenic effects, a series of novel 1, 3, 5-thiadiazine-2-thione derivatives bearing a hydrazide moiety were designed and synthesized, and their structureswere confirmed by FT-IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR and HRMS. Antifungal bioassays in vitro indicated that most of target compounds displayed good selectivity and specificity aganist Rs relative to Fg, Bc and Cc. Strikingly, six title compounds 5b, 5c, 6d, 7b, 7c and 7d exhibited remarkable antifungal activities against Rs in vitro, with corresponding EC₅₀ values of 0.24, 0.44, 0.25, 0.28, 0.35 and 0.46 μg/mL, which are obviously better than carbendazim (0.55 μg/mL). The in vivo anti-Rs effects of title compound 5b as a key representative were further evaluated on rice leaves to investigate the application potentials of title compounds as agricultural fungicides. The in vivo anti-Rs control efficacies of title compound 5b, which reached 98.58% at 200 μg/mL and 61.27% at 100 μg/mL, indicate that 1, 3, 5-thiadiazine-2-thione derivatives bearing a hydrazide moiety may have the research value of further structural optimization.

  Acknowledgments

  The authors gratefully acknowledge the grants from the National Natural Science Foundation of China (No. 31772209) and the Fundamental Research Funds for the Central Universities of China (No. KYTZ201604).

  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.2019.03.038.

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• 

  Figure 1  Design strategy of title compounds.

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  Scheme 1  Synthesis route of title compounds.

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  Figure 2  In vivo anti-Rs effect photographs of bioactive compounds on rice leaves. (A) Blank control; (B) 5b at 100 μg/mL; (C) 5b at 200 μg/mL; (D) Hymexazol at 200 μg/mL; (E) Carbendazim at 100 μg/mL.

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  Table 1.  In vitro antifungal EC₅₀ values (μg/mL) of title compounds 5–7.^a

  下载: 导出CSV

  Table 2.  In vivo anti-Rs control efficacies of bioactive compounds on rice leaves.^a

  下载: 导出CSV
•