1. Introduction

Tobacco mosaic virus (TMV), a positive-sensesingle stranded RNA virus, infects a wide range of plants, especially tobacco, vegetable and other members of the Solanaceae family. The virus is globally ubiquitous and causes great crop loss [1]. Therefore, TMV is an uncontrollable form of "plant cancer". Ribavirin and Ningnanmycin are common treatments for TMV infections under field conditions. However, Ribavirin has poor inactivation activity, and Ningnanmycin unstable and expensive [2, 3]. Therefore, the development of a novel, simple, and high-efficiency antiviral agents is a significant challenge in pesticide science [4].

Chalcone is a natural product, that exists in licorice, saffron and other medicinal plants. Chalcones have a wide range of pharmacological activities [5], such as anti-Alzheimer's disease [6], antitumor [7], antibacterial [8], insecticidal [9], anti-inflammatory [10], anti-oxidation [11], antiplatelet [12], and antiviral activities [13-15]. In our previous work, we reported a variety of chalcone derivatives and evaluated for their antiviral activity [16-18]. The results indicated that these compounds have moderate to good antiviral activity against TMV.

1, 3-Dichloropropene is a water-soluble and volatile compound that is mainly used as a pre-planting fumigant and nematicide. It is widely used in the US and other countries [19]. Its analogue, 1, 1-dichloropropene, also displays similar activities [20-23]. Pyridalyl, a commercially available 3, 3-dichloro-2-propenyloxy-substituted compound, has excellent controlling effects on various lepidopterous and thysanopterous insects. It shows no cross-resistance with the existing insecticides, such as synthetic pyrethroids, organic phosphates, nicotinic insecticides and ryanodine insecticides [24]. 1, 1-Dichloropropene compounds have drawn scientific attention as an interesting foundation for generating new lead compounds. Recently Liu et al. [25] synthesized several dichloroallyloxy-phenol derivatives by introducing various substituted phenyl groups into the pyrazole ring. The resulting insecticidal compounds are active against P. xylostella at 6.25 μg/mL and are more potent than the reference pyridalyl. In 2015, Yang and coworkers synthesized a series of the novel 1, 1-dichloropropene derivatives bearing diverse heterocycles with insecticidal activity against bollworm and P. litura [26]. Though, the 1, 1-dichloropropene group is an important and applicable functional group for designing the insecticide, the corresponding derivatives that contain the 1, 1-dichloropropene moiety with antiviral activities have not been reported.

In order to evaluate antiviral activity of 1, 1-dichloropropene compounds, the novel chalcone derivatives that contain the 1, 1-dichloropropene moiety were designed and synthesized. The physical characteristics were determined by ¹H NMR, ¹³C NMR, IR and elemental analysis, and their antiviral activities were evaluated. The bioassay results indicated that the target compounds exhibited moderate to good inactivation activity against TMV. Compounds 7g, 7h, 7m, 7n, 7w, and 7z showed higher antiviral activity against TMV with EC50 values of 52.9, 45.6, 52.2, 53.8, 50.6, and 53.4 μg/mL, respectively, compared with Ribavirin (145.1 μg/mL). Furthermore, the interactions between target compounds and tobacco mosaic virus coat protein (TMV CP) were investigated by the fluorescence spectroscopy and microscale thermophoresis (MST).

2. Results and discussion 2.1. Chemistry

The synthetic route of chalcone derivatives that contain the 1, 1-dichloropropene moiety is summarized in Scheme 1. Intermediate 1 can be easily prepared with 1, 1, 1, 3-tetrachloropropane [21]. Hydroquinone, 1, 1, 3-trichloroprop-1-ene 1 and K₂CO₃ were refluxed in CH₃CN for 5 h to obtain intermediate 2 after solvent removal and dichloromethane extraction. Intermediate 2 and diethylamine as a catalyst were stirred in toluene. The mixture was slowly added to a solution of SO₂Cl₂. Then, the reaction mixture was stirred at 56 ℃ for 5 h to produce intermediate 3 [27]. Intermediate 3, K₂CO₃, KI, and 1, 3-dibromopropane were stirred at room temperature for 12 h to yield intermediate 4. Intermediates 5 and 6 were prepared in accordance with previously reported methods [25, 26]. Finally, intermediates 4 and 6 were reacted by etherification to yield the target compounds 7a-7z. The target products were characterized by ¹H NMR, ¹³C NMR, IR, and elemental analysis. The data of spectroscopic characterization of the target compounds can be found in the Supporting Information.

2.2. Antiviral activity

The antiviral activities against TMV of the title compounds 7a-7z were determined via the half-leaf method [28, 29]. As shown in Table 1, the bioassay results indicated that all of the target compounds exhibited moderate to good activity against TMV at the concentration of 500 μg/mL. Among the targets, compounds 7g, 7h, 7m, 7n, 7w, and 7z showed better inactivation activity against TMV, with the inhibition rates of 93.1%, 94.8%, 93.5%, 92.9%, 94.0%, and 93.1%, respectively. These inhibition rates were similar to that of Ningnanmycin (94.1%) and superior to that of Ribavirin (72.9%). The other compounds displayed moderate inactivation activity against TMV.

To establish the structure-activity relationships of the target compounds, the EC₅₀ values of the target compounds' inactivation activities against TMV were evaluated. The evaluation results are presented in Table 2. All target compounds displayed excellent inactivation activity. Compounds 7g, 7h, 7m, 7n, 7w, and 7z showed antiviral activity against TMV with EC₅₀ and EC₉₀ values of 52.9, 45.6, 52.2, 53.8, 50.6, 53.4 and 332.9, 327.5, 331.5, 333.7, 331.0, 333.2 μg/mL, respectively. The inactivation activity of these compounds was better than that of Ribavirin (145.1 and 793.1 μg/mL). Among these six compounds, compound 7h exhibited the best inactivation activity against TMV and was similar to that of Ningnanmycin (46.9 and 329.4 μg/mL). Structureactivity relationship analysis revealed that differences in R groups have an important influence on inactivation activity. When the R groups were 4-OCH₃-Ph(7h), 3-OCH₃-Ph (7m), 4-OCH₂CH₃-Ph (7q), and 4-OCF₃-Ph (7z), the corresponding compounds displayed good activity compared with 7e (2-Cl-Ph), 7f (2-Br-Ph), 7g (2-F-Ph) and 7k (2-CF₃-Ph). Therefore, the introduction of electrondonating groups improves the anti-TMV activity of the chalcone compounds.

2.3. Interaction between 7h and TMV-CP

Fluorescence spectroscopy and MST analysis were used to investigate the interactions between target compounds and TMV CP [30]. The test methods are shown in the supporting data. The fluorescence spectrum showed that the binding constant K_a between 7h and TMV-CP was 2.63 ×10⁵ L/mol, which was similar to that of Ningnanmycin (2.39 ×10⁵ L/mol) and superior to that of Ribavirin (2.57 ×10³ L/mol). Compound 7r (3.98 × 10⁴ L/mol) showed moderate affinity and 7j (6.81 ×10³ L/mol) showed weak affinity, as shown in Fig. 1 and Table 3. To further confirm the results of fluorescence spectroscopy, the K_d between compound 7h and TMV CP was investigated with MST. The result of MST analysis was consistent with the data presented by the fluorescence spectrum, as shown in Fig. 2 and Table 3. The K_d between 7h and TMV-CP was 9.51 μmol/L, which was higher than those of 7r (34.8 μmol/L) and 7j (162 μmol/L). Therefore, the result showed that compound 7h strongly interacted with TMV CP.

3. Conclusion

In summary, we synthesized novel chalcone derivatives that contain a 1, 1-dichloropropene moiety. The anti-TMV activity of the target compounds 7a-7z was evaluated with the in vivo half-leaf method. Bioassay results showed that the target compounds were better than Ribavirin (145.1 μg/mL) against TMV. In particular, compound 7h possessed excellent inactivation activity against TMV and had an EC₅₀ value of 45.6 μg/mL, which was similar to that of Ningnanmycin (46.9 μg/mL). In addition, fluorescence spectroscopy and MST showed that compound 7h strongly interacted with TMV CP. Further studies on the mechanism of inactivation are currently underway.

4. Experimental 4.1. Synthesis

Unless otherwise stated, allreactants andreagents were purchased from commercial suppliers. The melting points were determined on an XT-4 binocular microscope (Beijing Tech Instrument, China) and were not corrected. Mass Spectra were obtained with a LC-MS 1100/MSD spectrometer (Agilent company, America). ¹H NMR and ¹³C NMR (solvent CDCl₃) spectral analyses were performed on a JEOL ECX 500 NMR spectrometer operated at 500 MHz for ¹H NMR, 125 MHz for ¹³C NMR at room temperature and TMS as the internal standard. Elements were performed with an Elemental Vario-Ⅲ CHN analyzer, and IR spectra were acquired in KBr on a Bruker VECTOR 22 spectrometer. Analytical thin layer chromatography (TLC) was performed on silica gel GF₂₅₄.

General procedure for the preparation of the key intermediate 6a-6z: Intermediates 6a-6z were prepared by performing previously reported synthetic procedures [24, 25]. A mixture of 4-hydroxychalcone (1.00 g, 4.2 mmol) and nitromethane (5.12 g, 83.9 mmol) in anhydrous ethanol was stirred. The mixture was refluxed for 2 h after the addition of KOH (0.28 g, 5.04 mmol). The reaction was monitored to completion via TLC. After the reaction system was cooled, its pH was adjusted with acid. Then, the mixture was filtered and the supernatant was discarded. The crude solid was purified via TLC on silica gel (petroleum ether/ethyl acetate = 3:1). The physical and chemical properties and spectroscopic characterization of the target compounds can be found in the Supporting information.

General synthetic procedures for the title compounds 7a-7z: A mixture of intermediates 4 (1.2 mmol), 6 (1.0 mmol) and potassium carbonate (2.0 mmol), with KI in acetone as the catalyst, was stirred and refluxed for 8 h. The solvent was evaporated in vacuo and the residue was isolated via column chromatography on silica gel (petroleum ether/dichloromathane = 1:1) to obtain the title compounds. The physical and chemical properties and spectroscopic characterization of the target compounds can be found in the Supporting information.

4.2. Bioassay

The in vivo antiviral activities against TMV of the target compounds were examined. Ningnanmycin was used as the control. EC₅₀ of the antiviral activity against TMV was determined. The assays were repeated thrice [28, 29].

4.3. K_d of compound 7h to TMV CP

The in vitro binding affinity of the title compounds on TMV-CP was performed by MST and fluorescence spectroscopy measurement as previously described [30].

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Nos. 21362004, 21562013) and Subsidy Project for Outstanding Key Laboratory of Guizhou Province in China (20154004).

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.2017.03.013.