Design, synthesis and biological activities of echinopsine derivatives containing acylhydrazone moiety

Based on the broad-spectrum biological activities of echinopsine and acylhydrazones, a series of echinopsine derivatives containing acylhydrazone moieties have been designed, synthesized and their biological activities were evaluated for the first time. The bioassay results indicated that most of the compounds showed moderate to good antiviral activities against tobacco mosaic virus (TMV), among which echinopsine (I) (inactivation activity, 49.5 ± 4.4%; curative activity, 46.1 ± 1.5%; protection activity, 42.6 ± 2.3%) and its derivatives 1 (inactivation activity, 44.9 ± 4.6%; curative activity, 39.8 ± 2.6%; protection activity, 47.3 ± 4.3%), 3 (inactivation activity, 47.9 ± 0.9%; curative activity, 43.7 ± 3.1%; protection activity, 44.6 ± 3.3%), 7 (inactivation activity, 46.2 ± 1.6%; curative activity, 45.0 ± 3.7%; protection activity, 41.7 ± 0.9%) showed higher anti-TMV activity in vivo at 500 mg/L than commercial ribavirin (inactivation activity, 38.9 ± 1.4%; curative activity, 39.2 ± 1.8%; protection activity, 36.4 ± 3.4%). Some compounds exhibited insecticidal activities against Plutella xylostella, Mythimna separate and Spodoptera frugiperda. Especially, compounds 7 and 27 displayed excellent insecticidal activities against Plutella xylostell (mortality 67 ± 6% and 53 ± 6%) even at 0.1 mg/L. Additionally, most echinopsine derivatives exhibited high fungicidal activities against Physalospora piricola and Sclerotinia sclerotiorum.

Plant virus diseases can be caused by more than 900 viruses, which reduce grain production and lead to huge economic losses all over the world [1][2][3] . As a well-studied plant virus, tobacco mosaic virus (TMV) belongs to single-stranded RNA virus of the family togaviridae 4 and it can infect 268 species of plants in 38 families, such as tobacco, tomato, pepper, cucumber, causing their leaves to grow spots, wither and even leading to yield reduction [5][6][7] . Although commercially available plant virus inhibitors ningnanmycin and ribavirin are widely used to control TMV, their inhibitory effects are lower than 60% 8 . Thus, the development of efficient alternative TMV inhibitors is still in great request.
Natural products are an important source of plant virus inhibitor discovery. Compared with traditional synthetic plant virus inhibitor, plant virus inhibitor derived from natural products have many advantages, including low toxic, environmentally friendly, easy to decompose and specific to target species, etc 9,10 . Song et al. reported that the EC 50 value of purine nucleoside derivative for the inactivating activity against TMV was 48 mg/L, which was better than that of ningnanmycin (88 mg/L) 11 . Li et al. first found that phenanthroindolizidine alkaloid, (R)-antofine, exhibited a good inhibitory effect against TMV 12 . Wang et al. found some β-carboline analogues 7 , hemigossypol 13 , dehydrobufotenine derivatives 14 , pityriacitrin marine alkaloids 15 , pulmonarin alkaloids 16 and hamacanthin derivatives 17 exhibited higher anti-TMV activities than ningnanmycin. Many other natural alkaloids derivatives were also developed as potential TMV inhibitors [18][19][20][21][22][23][24][25][26][27] . Although a variety of natural product derivatives have been found to exhibit high anti-TMV activity, few of them have been applied successfully in agriculture. Thus, it is necessary to discover novel natural TMV inhibitors with diverse structures.
Echinopsine is a quinoline alkaloid isolated from Echinops sphaerocephalus L., the root of which was used as traditional Chinese medicine for treatment of deep-rooted breast carbuncles, ulcer, sodoku and breast milk stoppage. Although the bioactivity of Echinops sphaerocephalus L. extract has been widely studied 28 , the biological activity of echinopsine is still not clear. The anti-TMV activity of echinopsine has not been reported so far. However, a variety of natural alkaloids containing echinopsine moiety showed herbicidal, insecticidal, bactericidal, www.nature.com/scientificreports/ anti-tumor, antifungal and antifeedant activities, etc. (Fig. 1) 29 , indicating echinopsine moiety has potential broad-spectrum biological activities. Based on this, the anti-TMV activity of echinopsine was investigated by our group and the result shows that the inactivation, curative and protection activities of echinopsine (49.5 ± 4.4%, 46.1 ± 1.5% and 42.6 ± 2.3% at 500 mg/L, in Table 1) were higher than that of ribavirin (38.9 ± 1.4%, 39.2 ± 1.8%, 36.4 ± 3.4%, at 500 mg/L). The biological activities of acylhydrazone compounds have always been the focus of pharmacological research [30][31][32][33] . Variety of compounds with acylhydrazone functional group (−CONHN=) showed good bactericidal, herbicidal or insecticidal activities, such as benquinox 34 , saijunmao 35 , metaflumizone 36 and diflufenzopyr 37 . Based on the high biological activities of echinopsine and acylhydrazone structure, in order to find echinopsine derivatives with higher anti-TMV activities and summarize their structure-activity relationship, a series of echinopsine derivatives containing acylhydrazone moieties were designed, synthesized and characterized in this work (Fig. 2). Their anti-TMV activities were studied for the first time. Besides, in order to see if these compounds have broad spectrum bioactivity, their insecticidal and fungicidal activities were also investigated.

Materials and methods
Instruments. 1 H NMR spectra were obtained at 400 MHz using a Bruker AV400 spectrometer in CDCl 3 or DMSO-d 6 solution with tetramethylsilane as the internal standard. HRMS data were obtained on an FTICR-MS instrument (Ionspec 7.0 T). The melting points were determined on an X-4 binocular microscope melting point apparatus without correction.
Biological assay. The anti-TMV, insecticidal and fungicidal activities of the synthesized compounds were tested using our previously reported methods 38,39 and the methods can also be found in the "Supporting Information SI".
General synthesis. Ribavirin (Topscience Co., Ltd.), chlorothalonil (Bailing Agrochemical Co., Ltd.), carbendazim (Bailing Agrochemical Co., Ltd.) and other reagents were purchased from commercial sources and used as received. All anhydrous solvents were dried and purified according to standard techniques. The synthetic routes were given in Fig. 3. Echinopsine was prepared according to literature 40 .    General procedure for the preparation of compounds 1-27. To a round bottomed flask (100 mL)

Synthesis of 1-methyl-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (C). Compound
were added methanol (50 mL), compound C (3 mmol), one benzaldehyde from D 1 -D 27 (3 mmol) and p-methylbenzene sulfonic acid (0.6 mmol). The reaction suspension was refluxed for 8 h. The reaction suspension was cooled to room temperature and partial methanol was evaporated under reduced pressure until a large amount of precipitation precipitated. The precipitate was filtered and washed several times with cool methanol to afford compounds 1-27. Data for compounds 1-27 can be found in the "Supporting Information SI".

Results and discussion
Synthesis. The preparation of compound B was carried out according to literature 41 (Fig. 3). Acetonitrile was used as solvent instead of DMF and the reaction was accomplished in 91.2% yield. Then product B reacted with hydrazine hydrate under reflux to afford hydrazine C, which can react subsequently with aldehyde D 1 -D 27 to give hydrazine 1-27 as products in 52.7-95.3% yields. During the synthesis of acylhydrazone 1-27, only trans isomers were obtained, which may due to the fact that trans isomers are more stable than cis isomers thermodynamically. Compounds 1-27 can precipitate from methanol, which made the purification of acylhydrazone derivatives easy and suitable for large-scale production.
Fungicidal activity. The fungicidal results of compounds 1-27 and echinopsine are listed in Table 4. The commercial fungicide carbendazim and chlorothalonil were used as positive control. Overall, echinopsine and their derivatives exhibited broad-spectrum fungicidal activities against 14 kinds of phytopathogenic fungi. Most compounds showed relatively high fungicidal activities for Physalospora piricola and Sclerotinia sclerotiorum, among which the fungicidal activities of compounds 1-14 containing substituted phenyl were relatively higher than compounds 15-26 containing heterocyclic rings. Compound 13 and 14 showed more than 50% inhibitory rate against five and six fungi respectively. Compound 2 showed the widest spectrum of fungicidal activity, with more than 60% inhibitory rate against eight fungi. Compound 7 exhibits 89.0 ± 1.9% inhibitory rate against Rhizoctonia cerealis at 50 mg/L, higher than carbendazim and chlorothalonil. In summary, a series of novel echinopsine derivatives containing acylhydrazone moieties were designed, synthesized and their antiviral, insecticidal, and fungicidal activities were studied. The bioassays results showed that most compounds exhibited moderate to good anti-TMV activities in vivo, among which echinopsine (I) and its derivatives 1, 3, 7 showed higher anti-TMV activities than those of ribavirin, which can be used as lead structures for the development of anti-TMV drugs. Some compounds exhibited moderate to good insecticidal activity to P. xylostella, M. separata and S. frugiperda. In addition, most of these compounds exhibited good fungicidal activities against P. piricola and S. sclerotiorum. Further investigation on structural optimization and the mechanism of action are in progress in our laboratory.  www.nature.com/scientificreports/    www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.