English

• 1.   Introduction

  Heterocyclic compounds, such as thiazole, pyrazole, pyridine, and triazole, play important roles in medical chemistry and agrochemicals [1]. 1, 2, 3-Thiadiazole, one typical heterocycle widely used in drug discovery, has increasingly attracted attention thanks to its versatile biological activities, such as anti-HIV, antitumor, antiviral, systemic acquired resistance, insecticidal, fungicidal, and herbicidal activities [2–14]. For instance, 4-methyl-1, 2, 3-thiadiazole derivatives Ⅰ (Fig. 1) was an excellent necroptosis inhibitor (EC₅₀ = 1.0 mmol/L) [14]. Meanwhile, Fan reported a series of 4-methyl-1, 2, 3-thiadiazole derivatives containing 1, 2, 4-triazolo [3, 4-b][1, 3, 4]thiadiazoles showed good fungicidal activity, especially compound Ⅱ (Fig. 1) against Rhiz octonia solani and Pellicularia sasakii with EC₅₀ values of 4.1 and 2.5 μg/mL, respectively [15]. Tiadinil (Fig. 1), a novel fungicide commercialized containing 1, 2, 3-thiadiazole, also had good induction activity against tobacco mosaic virus (TMV) [2].

  图 1

  

  图 1  The chemical structures of compounds containing 1, 2, 3-thiadiazole.

  Figure 1.  The chemical structures of compounds containing 1, 2, 3-thiadiazole.

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  Carbohydrazide hydrazone is an attractive and versatile scaffold with relevant application in several areas, including asymmetric catalysis, coordination chemistry, agrochemicals and pharmacology [16–18]. A number of carbohydrazide hydrazone derivatives possess interesting bioactivity such as antifungal, anti-inflammatory, antiplatelets, antimalarial and anticancer activities (Fig. 2) [18–22]. Metaflumizone, Diflufenzopyr, and Benquinox (Fig. 2) are known as the commercial agrochemicals containing this scaffold [16]. In view of superior properties and with the aim to find new lead compounds with high biological activity, we introduced carbohydrazide hydrazone into the structure of Tiadinil to replace the amide linkage, and designed, synthesized a series of novel 4-methyl-1, 2, 3-thiadiazole-5-carboxaldehyde benzoyl hydrazone derivatives (Fig. 3). All the compounds were evaluated for the activities against six plant fungal pathogens in vitro and tobacco mosaic virus in vivo (curative activity). Moreover, the initial structure-activity relationship (SAR) of these compounds was also analysized in the present work. The results might be valuable for the discovery of eco-friendly agrochemicals.

  图 2

  

  图 2  The chemical structures of compounds containing carbohydrazide hydrazone.

  Figure 2.  The chemical structures of compounds containing carbohydrazide hydrazone.

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  图 3

  

  图 3  Design strategy of the target compounds.

  Figure 3.  Design strategy of the target compounds.

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  2.   Results and discussion

  2.1   Chemistry

  The synthetic route for the title compounds 8a–8x was illustrated in Scheme 1. The key intermediate 4-methyl-1, 2, 3-thiadiazole-5-carboxaldehyde 6 was synthesized from dimethyl carbonate via five steps including hydrazidation, condensation, cyclization, reduction and oxidation by conventional synthetic methods from commercially available starting materials. Then the target compounds 8a–8x were easily obtained in 60%–96% yields through addition-elimination condensation between compound 6 and different substituted benzoyl hydrazine 7. Their structures were fully characterized by melting points, ¹H NMR, ¹³C NMR, IR spectra and elemental analysis (Supporting information).

  Scheme1

  

  图 Scheme1  Synthetic route of the title compounds 8a–8x.

  Scheme1.  Synthetic route of the title compounds 8a–8x.

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  In order to determine the stereo structure of the target compounds, a single crystal of representative compound 8g (R = 4-Br) was prepared by slow evaporation of a solution of compound 8g in ethanol at 4 ℃. As shown in Fig. 4, it gives a perspective view of 8g with the atomic labeling system, and the result demonstrates the —CH＝N—NH— bond bears an (E) rather than (Z) conformation. Moreover, the crystal is distribution with dimer form and there is an intermolecular hydrogen bond between the two subunits. The crystal conformation is more stable owing to the lower energy of the system. The crystal data for 8g: triclinic, a = 9.5767(5) Å, b = 9.6459(7) Å, c = 13.7997(9) Å, α = 84.771(6)°, β = 87.927(5)°, γ = 88.623(5)°, V = 1268.34(14) Å3, T = 104.6, space group P-1 (no. 2), Z = 4, μ(Mo Kα) = 3.398 mm^-1, D_calc = 1.703 g/cm³, μ= 3.398 mm^-1, 10125 reflections measured(6.08° ≤ 2Θ ≤ 52°), 4969 unique (R_int = 0.0263) were used in all calculations. The final R₁ was 0.0378 (I > 2σ(I)) and wR(F₂) was 0.0935 (all data). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre, and the deposition number was CCDC 1481606-1481607.

  图 4

  

  图 4  The X-ray crystal structure of 8g.

  Figure 4.  The X-ray crystal structure of 8g.

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  2.2   In vitro antifungal activity

  All the target compounds were evaluated for their fungicidal activities against six plant fungal pathogens. The results were shown in Table 1. The data suggested that all compounds had moderate to strong fungicidal activity. Compounds 8c, 8d, 8f, 8g, 8i, 8j, 8k, 8m, and 8n exhibited more than 90% inhibition activities against V. mali, similar to Tiadinil (92.9%) at 50 μg/mL. Compound 8k also displayed good activity against B. cinerea (87.5%), which is comparable with that of Tiadinil (80.4%) and Difenoconazole (92.1%). Unfortunately, most compounds did not show better activities for the rest plant fungal pathogens compared with Tiadinil and Difenoconazole. However, compounds 8k and 8n showed above 50% inhibition activity against other four fungi P. aphanidermatum, R. solani, F. moniliforme and A. solani. These results indicated that the title compounds are exclusively efficient against V. mali mycelial growth in vitro but not potent enough against the other five fungi. Thus, V. mali was chosen as the target for further study.

  表 1

  

  表 1  The in vitro antifungal activities of title compounds 8a–8x.

  Table 1.  The in vitro antifungal activities of title compounds 8a–8x.

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  In order to intensively study the structure and activity relationship of the compounds against V. mali, therefore, different concentrations of compounds 8a–8x were treated against V. mali. The EC₅₀ values were calculated using linear-regression analysis, and the results were shown in Table 1. Among these 24 compounds, 8d, 8f, 8g, 8i, 8j, and 8n showed lower EC₅₀ values than the commercial fungicide Tiadinil, especially 8g and 8n with the EC₅₀ values of 1.6 and 1.9 μg/mL, respectively, which were 4-fold higher potency than Tiadinil. The results proved that introducing carbohydrazide hydrazone into the structure of Tiadinil to replace the amide linkage is favorable to improve the fungicidal activity. However, the compounds showed weaker effectiveness than Difenoconazole.

  Structure-activity relationship analysis generally indicates that: (ⅰ) The position of the substituent R on the benzene ring played an important role in fungicidal activities: meta-position exhibited weaker activity than the para-position, however, better than the ortho-position. Taking chloro substituents (8h–8j) as an instance, it is obvious that 8j (R = 4-Cl, 2.8) < 8i (R = 3-Cl, 3.8) < 8h (R = 2-Cl, 19.4) (" < " means inferior EC₅₀ values). The 2, 5-dichloro substituent 8l (36.9 μg/mL) showed the poorest activity, which is even weaker than 8h. The 2, 4-dichloro substituent 8k (8.2 mg/ mL) showed the moderate activity, which is much higher than 8h and 8l, but lower than 8j. The same rule can also be found in 8b–8d (-F), 8e–8g (-Br), 8m–8n (-CF₃), 8o–8q (-NO₂) and 8r–8s (-CH₃) groups. (ⅱ) The electron-withdrawing groups are more favorable to the activity than the electron-donating groups in the same position. For example, when substituent R is in the para position, compounds with the electron-withdrawing (8d, 8g, 8j, 8n, and 8q, ) are more potent than that with electron-donating substituent (8s–8u). Among of them, except 8q (4-NO₂, 10.2 mg/ mL) is close with 8s (4-CH₃, 10.9 μg/mL) and 8t (4-tBu, 9.4 mg/ mL), and the other four compounds 8d (4-F, 7.1 μg/mL), 8g (4-Br, 1.6 μg/mL), 8j (4-Cl, 2.8 μg/mL) and 8n (4-CF₃, 1.9 μg/mL) are much higher than that of 8s and 8t. In conclusion, the compound containing halogen at para position exhibits the best activity.

  Different from most compounds with a very narrow spectrum against the fungi tested in Table 1, compound 8k showed broad spectrum and thus was selected to determine the EC₅₀ values against the six fungi. As illustrated in Table 2, 8k displayed different levels activity against V. mali, B. cinerea, P. aphanidermatum, R. solani, F. moniliforme and A. solani with EC₅₀ values of 8.20, 24.42, 15.80, 40.53, 41.48 and 34.16 μg/mL, respectively, which are still weaker than positive control except P. aphanidermatum.

  表 2

  

  表 2  The EC₅₀ values of compound 8k against six fungal pathogens.

  Table 2.  The EC₅₀ values of compound 8k against six fungal pathogens.

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  2.3   In vivo antivirus activity

  Curative activity of all title compounds against TMV in vivo was evaluated. The results were shown in Table 3, in which all the compounds were active against TMV to some extent at the tested concentration. Among them, compounds 8a, 8b, 8e, 8j, 8m, 8o, and 8u presented moderate curative activity at 500 μg/mL, which were higher than that of standard Tiadinil. Especially, compounds 8a and 8b showed good anti-TMV activity with inhibition activity of 47.2% and 49.9%, respectively, which were close to the positive control Ningnanmycin (57.9%). Interestingly, compounds containing R groups at ortho-position of phenyl have higher activity than those R groups at meta-position. The results provide useful clues for further discovery of novel lead structures possessing good antivirus activity.

  表 3

  

  表 3  The curative effects of test compounds against TMV in vivo (500 μg/mL).

  Table 3.  The curative effects of test compounds against TMV in vivo (500 μg/mL).

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  3.   Conclusion

  A series of novel 4-methyl-1, 2, 3-thiadiazole-5-carboxaldehyde benzoyl hydrazone derivatives were designed and synthesized through six steps with dimethyl carbonate as starting material. The bioassay indicated that the title compounds exhibited moderate to strong antifungal activities against six fungi in vitro at 50 μg/mL and presented a certain degree of curative activity against TMV at 500 μg/mL. Compounds 8g and 8n exhibited outstanding activities against V. mali with the EC₅₀ values of 1.64 and 1.87 μg/mL, respectively, which are 4-fold higher potency than the control Tiadinil. Moreover, compound 8k showed broad spectrum activities against V. mali, B. cinerea, P. aphanidermatum, R. solani, F.moniliforme and A. solani with EC₅₀ values of 8.20, 24.42, 15.80, 40.53, 41.48, and 34.16 μg/mL, respectively. This result indicated that replacing the amide linkage of Tiadinil with carbohydrazide hydrazone is favorable to improve the biological activity of lead compound. The title structure can be a new lead for further modification.

  4.   Experimental

  4.1   Synthesis

  Melting points of all compounds were determined on an X-4 binocular microscope (Fukai Instrument Co., Beijing, China), with an uncorrected thermometer. ¹H NMR spectra and ¹³C NMR spectra were recorded on Bruker AM-300 (300 MHz, 75 MHz) spectrometer with CDCl₃ as the solvent and TMS as the internal standard. IR spectra were recorded on a Perkin Elmer Spectrum 47 100 FT-IR spectrometer with a KBr disk. Elemental analysis data were carried out on a Vario EL Ⅲ elemental analyzer. Single-crystal X-ray diffraction was measured on a Gemini E single-crystal diffractometer. All the reagents were obtained commercially and used after further purification. Column chromatography purification was carried out by using silica gel (Merck 60, 200–300 mesh).

  Synthesis of intermediates 2–6 were according the references [5, 11], the general procedures are given in the Supporting information. General procedures for target compounds 8a–8x: To a solution of corresponding benzoyl hydrazine 7 (3 mmol) in anhydrous ethanol (20 mL), an intermediate 6 (0.39 g, 3 mmol) was added. The resulting mixture was then stirred at refluxed temperature, when TLC analyses indicated the disappearance of the starting material, then cooled to room temperature, followed by filtered to obtain the solid. The solid was recrystallized in ethanol to give corresponding target compounds 8.

  4.2   Bioassays

  Antifungal activity (in vitro): The fungicidal activity was evaluated according to the mycelium growth rate method in the references at the concentration of 50 μg/mL [23, 24]. Fungi used in this study included six plant fungal pathogens (Valsa mali, Botrytis cinerea, Pythium aphanidermatum, Rhizoctonia solani, Fusarium moniliforme and Alternaria solani). Tiadinil and Difenoconazole were used as standard and positive controls, respectively. The detailed procedures were listed in the Supporting information.

  Antivirus activity (in vivo): The leaves of N. tabacum L. growing at the same ages were selected. TMV at a concentration of 0.06 μg/mL was dipped and inoculated on the whole leaves. After the leaves were dried in the greenhouse, the compound solution was smeared on the left side, and the solvent was smeared on the right side for control. The local lesion numbers were then recorded 3–4 d after inoculation. For each compound, three repetitions were conducted. All compounds were tested at 500 μg/mL. Tiadinil and ningnanmycin were used as standard and positive controls, respectively [25].

  The activity data of curative effects against TMV was calculated by the following equation:

  where: Y is the antivirus inhibition ratio (%), CK is the number of viral inflammations on the control leaves in vivo, and A is the number of viral inflammations on the target compound treated leaves in vivo.

  Acknowledgments

  The authors are grateful to Professor Xi-Li Liu at College of Plant Protection, China Agricultural University, for her kind help for supplying the fungi. We thank the financial support from the National Natural Science Foundation of China (No. 21132003).

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.02.002.

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  Figure 1  The chemical structures of compounds containing 1, 2, 3-thiadiazole.

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  Figure 2  The chemical structures of compounds containing carbohydrazide hydrazone.

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  Figure 3  Design strategy of the target compounds.

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  Scheme1  Synthetic route of the title compounds 8a–8x.

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  Figure 4  The X-ray crystal structure of 8g.

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  Table 1.  The in vitro antifungal activities of title compounds 8a–8x.

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  Table 2.  The EC₅₀ values of compound 8k against six fungal pathogens.

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  Table 3.  The curative effects of test compounds against TMV in vivo (500 μg/mL).

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