Natural phytoalexin stilbene compound resveratrol and its derivatives as anti-tobacco mosaic virus and anti-phytopathogenic fungus agents

Plant diseases caused by plant viruses and pathogens seriously affect crop yield and quality, and it is very difficult to control them. The discovery of new leads based on natural products is an important way to innovate pesticides. Based on the resveratrol is a kind of natural phytoalexin, but it cannot be used as candidate for the development of new drug due to its poor druggability. The phenolic hydroxyl groups in the resveratrol structure are easily destroyed by oxidation, in order to improve its stability, ester formation is the most commonly used modification method in drug design. Their structures were characterized by 1H NMR, 13C NMR and HRMS. The activity against tobacco mosaic virus (TMV) of these ester derivatives has been tested for the first time. The bioassay results showed part of the target compounds exhibited good to excellent in vivo activities against TMV. The optimum compounds III-2 (inhibitory rates of 50, 53, and 59% at 500 μg/mL for inactivation, curative, and protection activities in vivo, respectively), III-4 (inhibitory rates of 57, 59, and 51% at 500 μg/mL, respectively), and II-5 (inhibitory rates of 54, 52, and 51% at 500 μg/mL, respectively) displayed higher activity than commercial plant virucide ribavirin (inhibitory rates of 38, 37, and 40% at 500 μg/mL, respectively). Compounds I-9 and I-10 also showed excellent activities. The systematic study provides strong evidence that these simple resveratrol derivatives could become potential TMV inhibitors. The novel concise structure provides another new template for antiviral studies.

naturally occurring resveratrol (3,5,4′-trihydroxy-trans-stilbene, Fig. 1) is a phytoalexin which can be activated by adverse conditions of plants, protecting against fungal infections [4][5][6][7][8][9][10][11] . First isolated in 1940 from Veratrum grandiflorum by Takaoka 12,13 , and later, it has been obtained in larger quantities from the roots of Polygonum cuspidatum 14 , a plant used in traditional Chinese medicine. It attracted wider attention only in 1992 when its presence in wine was suggested as the explanation for cardioprotective effects 15 . Resveratrol is the most representative compound of stilbene analogues, resveratrol and its derivatives exhibits a wide range of intriguing biological activities, such as antibacterial 16 , antitumour 17,18 , antiviral 19 , antioxidant 20 , and antihypertensive activities 21 . Furthermore, stilbene moieties may have numerous agrochemical applications, such as in herbicides [22][23][24][25] . Recently, considerable attention has been focused on resveratrol derivatives. Although resveratrol possesses a series of pharmacological activities, its applications in the field of pesticide development have not been reported.
Studies have shown that modification of the structure of natural compounds can improve their biological activities. To improve the stability of resveratrol, many researchers have undertaken the synthesis and activity evaluation of resveratrol derivatives and analogues. They have modified the phenolic hydroxyl groups, double bonds and benzene ring of resveratrol so as to further understand the interactions among functional groups and its structure-activity relationship. Grow evidence indicates that this compound could be used as a lead compound in the design of drugs and resveratrol derivatives synthesized from the lead compound resveratrol or stilbene have a higher efficacy and lower toxicity [26][27][28][29] .
Whilst the antiviral activity of resveratrol has been extensively studied, little is known about the activity against plant viruses of resveratrol and resveratrol derivatives. Ester formation is the most commonly used modification method in drug design. A few resveratrol ester derivatives had been synthesized and tested for their antitumor activity. However, reports of the anti-TMV activity of the resveratrol ester derivatives are rather rare, and no examples are documented in the recent literature. In this paper, in order to increase the stability and druggability of resveratrol, we synthesized a series of resveratrol ester derivatives and their activities against TMV were evaluated (Fig. 2). The structure-activity relationships of these derivatives were discussed as well. Additionally, the synthesized derivatives were also investigated for their potential as fungicidal, or insecticidal agents. Resveratrol derivatives will become a research focus in future in agricultural applications.

Materials and methods
Instruments. All other commercial reagents and solvents were used as received without further purification. Reaction solvents were distilled from calcium hydride for dichloromethane and from sodium metal and benzophenone for tetrahydrofuran. E-resveratrol (98%) was purchased from Shanxi Sciphar Hi-tech Industry Co., Ltd. (Shanxi, China). Reaction progress was monitored by thin-layer chromatography on silica gel GF254 with detection by ultraviolet (UV). Melting points were obtained using an X-4 binocular microscope melting point (mp) apparatus and are uncorrected. Yields were not optimized. 1 H-NMR spectra and 13 C-NMR spectra were recorded utilizing a Bruker AV400 spectrometer with CDCl 3 as solvent and tetramethylsilane as internal standard. Chemical shifts (δ) were given in parts per million (ppm). High-resolution mass spectra (HRMS) data were obtained with a Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) spectrometer (ionspec, 7.0 T).    Biological assay. Each bioassay was repeated three times at 25 ± 1 °C. The activity results were estimated according to a percentage scale of 0-100 (0 indicating no activity and 100 indicating total mortality). The bioassay procedures for the anti-TMV, fungicidal, and insecticidal activities of the synthesized compounds are described in detailed in our published literature and can also be found in the "Supporting Information S1" 30 .

Results and discussion
Synthesis. In order to investigate structure-activity relationships (SARs), resveratrol was chosen as a precursor according to the in vivo anti-TMV activity listed in Table 1. Several series of resveratrol derivatives I-1-I-10, II-1-II-5 and III-1-III-4 were synthesized according to procedures in Scheme 1. As shown in Scheme 1, commercially available E-resveratrol was reacted with corresponding aryl chloride, alkyl chloride and heterocyclic chloride to give resveratrol esters I-1-I-10, II-1-II-5 and III-1-III-4 in good yields. The detailed procedure is given in "Materials and methods". It's a simple route for the preparation of resveratrol derivatives with favorable yield.  Table 1. To make a judgment on the antiviral potency of the synthesized compounds, the commercially available plant virucide ribavirin and lead compound resveratrol were used as the controls. All of the compounds were tested at both 500 and 100 μg/ mL.
The synthesized compounds showed similar or higher in vivo activity against TMV than the commercial plant virucide ribavirin. At the concentration of 500 μg/mL, resveratrol ester derivative containing meta-thienyl moieties III-2 displayed the best inhibitory effect of inactivation activity, curative activity, and protection activity with values of 50, 53, and 59%, respectively. Ribavirin as a control was studied at the same conditions with values of 38, 37, and 40%, respectively. In contrast to the experimental data, the results indicated that compound III-2 was more efficient than ribavirin in vivo activity against TMV. At the concentration of 100 μg/mL, III-2 displayed inactivation activity, 25%; curative activity, 20%; and protection activity, 28%; which is higher than that of ribavirin (inactivation activity, 11%; curative activity, 13%; and protection activity, 12%). Resveratrol ester derivative III-4 bearing furan groups also gave relatively higher activity (57, 59, and 51% at 500 μg/mL) than ribavirin. Especially the curative activity at 500 μg/mL of III-4 was higher than ribavirin and resveratrol, which indicated that the introduction of heterocycle might lead to the increase of inhibitory effect. But introduction of pyridyl (III-1) and ortho-thienyl (III-4) resulted in a decline of anti-TMV activity 27 . Compound II-5 with alkyl groups such as methyl resulted in an obvious improvement of anti-TMV activity which exhibited better curative and protection activities in vivo against TMV than ribavirin. More importantly, the inactivation activity of compound II-5 was much higher than ribavirin. But other derivatives bearing alkyl groups II-2, II-4, II-1, II-3 in series II showed similar or lower in vivo anti-TMV activity than ribavirin 31 . The introduction of aromatic substituents resulted in a decline of anti-TMV activity, however aromatic substituents of resveratrol with electron-donating groups such as methyl (I-9) or t-butyl (I-10) showed more excellent in vivo anti-TMV activity than ribavirin 27 . Fungicidal activity. The resveratrol and its derivatives were also evaluated for their fungicidal activities with the commercial fungicides chlorothalonil and carbendazim as the controls. Resveratrol and its derivatives all exhibited fungicidal activities to some extent against 14 kinds of plant pathogens (Fusarium oxysporum sp. cucumeris; Cercospora arachidicola Hori; Physalospora piricola; Rhizoctonia cerealis; Bipolaris maydis; Colletotrichum orbiculare; Fusarium moniliforme; Alternaria solani; Fusarium graminearum; Phytophthora infestans; Phytophthora capsici; Sclerotinia sclerotiorum; Botrytis cinerea; Rhizoctonia solani) by mycelial growth method ( Table 2). As shown in Table 2, target compounds exhibited broad spectrum fungicidal activities against 14 kinds of phytopathogenic fungi at 50 μg/mL. However, compared with commercial fungicides carbendazim and chlorothalonil, these derivatives were less potent.  Table 3. The results indicated that many compounds exhibited higher activities than resveratrol. Especially compounds II-3, III-3, and III-4 exhibited obviously higher activities against oriental armyworm (70%, 70%, 60% at 600 μg/mL) than resveratrol. However, though some compounds exhibited insecticidal activity on some species to some extent, the potency of these compounds as insecticide was not comparable with that of commercial insecticides. More modification on the structure should be conducted.

Conclusion
In summary, the natural product resveratrol provides an unparalleled source of inspiration for the screening of antiviral drugs. With resveratrol as lead compound by chemical modification, a series of resveratrol ester derivatives were designed and synthesized. Their activities against TMV were evaluated. The optimum compounds III-2, III-4, and II-5, I-9, I-10 displayed higher activity than commercial plant virucide ribavirin. This paper also discussed the anti-TMV activities and the structure-activity relationships of resveratrol ester derivatives, providing a reference for the development of new drugs. Resveratrol as important phytoalexin, its structure is simple and is a kind of lead compound which is extremely potential in the field of pesticide development, so the structure modification of resveratrol is a very valuable and meaningful research work.    Table 3. Insecticidal activities of the target compounds I-1-I-10, II-1-II-5 and III-1-III-4 (mortality a , percent). a Average of three replicates. All results are expressed as the mean ± standard deviation (SD). b Positive control.