Design , Synthesis , and Biological Activity of Novel penta-1 , 4-dien-3-one Derivatives Containing a H-phosphonate Scaffold

A series of penta-1,4-dien-3-one containing a H-phosphonate scaffold were designed and synthesized. The structures of all title compounds were determined by 1H-NMR, 13C-NMR, 31P-NMR, and HRMS. Bioassay results showed that several of the title compounds exhibited remarkable antibacterial and antiviral activities. Among these, compounds 3c and 3o exhibited substantial antibacterial activities against Xanthomonas oryzae pv. Oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). In addition, compounds 3c, 3f, and 3r showed remarkable curative activities against tobacco mosaic virus (TMV), with 50% effective concentration (EC50) values of 290.0, 234.0, and 373.6 μg/mL, respectively. These were superior to that of ningnanmycin (386.2 μg/mL). Compound 3r exhibited comparative protective activity against TMV, with an EC50 value of 291.1 μg/mL, which was better than that of ningnanmycin (297.1 μg/mL). Notably, the solubility of all title compounds improved relative to the lead compound curcumin. These results suggest that penta-1,4-dien-3-one containing a H-phosphonate scaffold may be considered as an activator for antibacterial and antiviral agents.


Introduction
Pathogenic bacteria, such as rice bacterial leaf blight and citrus canker caused by the pathogens Xanthomonas oryzae pv.oryzae (Xoo) [1,2] and Xanthomonas axonopodis pv.citri (Xac) [3,4], strongly restrain the agricultural output worldwide and are difficult to control in agriculture.Furthermore, vegetables are susceptible to infection with the tobacco mosaic virus (TMV), one of the most severe pathogenic viruses, which causes considerable crop loss [5][6][7].To date, the few commercially available bactericides and plant virucides, such as thiodiazole-copper (TC), bismerthiazol (BT), ningnanmycin, and ribavirin not only enhance resistance in the target pathogens, but are also detrimental for both the environment and plant health [8].Therefore, developing new antibacterial and antiviral agents remains an important task for the medical community.
Pesticides based on natural products show more advantages than synthesized chemicals, e.g., low toxicity, simple decomposition, unique modes of action, and environmental friendliness [9,10].Therefore, it is a development trend to search for designs and to synthesize pesticides based on natural products.As an important analog of curcumin isolated from turmeric, penta-1,4-dien-3-one possess numerous potential biological activities, including antibacterial [11], antiviral [12], antifungal [13], and antitumor [14] activities.In a previous study from our group, we synthesized a series of penta-1,4-dien-3-one derivatives, most of which exhibited excellent antibacterial and antiviral activities [11,15,16].
H-phosphonate and its derivatives possess a wide range of biological activities [17][18][19][20][21][22][23] and are therefore widely employed as plant virucides, bactericides, fungicides, herbicides, and plant growth regulators.Because the phosphorus-carbon bond of phosphonates is not susceptible to enzymatic degradation, it possesses more cell permeability, a lipophilic nature, and good physiological stability [24,25].Over the past 10 years, a number of papers have reported their synthesis and biological activities [26][27][28][29].H-phosphonate has attracted considerate attention in the field of pesticide application.
Motivated by the above-mentioned findings and to continue our efforts for developing highly agrochemicals, we introduced a H-phosphonates scaffold into penta-1,4-diene-3-one derivatives, which might generate novel curcumin derivatives with potent biological activities.Thus, 18 penta-1,4-diene-3-one derivatives containing a H-phosphonate scaffold were designed, synthesized and evaluated for their antibacterial activities against Xac and Xoo in vitro and their antiviral activity against TMV in vivo.

Chemistry.
The synthetic route to penta-1,4-diene-3-one derivatives containing a H-phosphonate moiety is schematized in Scheme 1.According to previous reports [13,30], 4-hydroxybenzaldehyde and acetone were used as the starting materials, and reacted with 5% NaOH for 12 h at normal temperature to obtain the intermediate (E)-4-(4-hydroxyphenyl)but-3-en-2-one (1).Intermediate 2 was obtained via condensation of intermediate 1 with substituted aldehydes.Finally, the title compounds 3a-3r were synthesized via substitution of intermediates 2 and H-phosphonate with Et3N in CCl4 at normal temperature for 24 h.
The structures of all title compounds were determined by 1 H-NMR, 13 C-NMR, 31 P-NMR, and HRMS, and the spectra data are shown in the Supporting Information.The representative data for 3a are shown below.In 1 H NMR spectra, multiplet signals at δ 7.70-6.99ppm indicate the presence of protons in olefinic bonds and aromatic nucleuses, and a singlet at δ 3.87-3.89ppm indicates the presence of -CH3 groups.Absorption signals at δ 188 and 55 ppm in 13 C NMR spectra confirm the presences of -C=O-and -CH3 groups, respectively.The HRMS spectra of target compounds show characteristic absorption signals of [M+H] + ions, which is consistent with their molecular weight.
a The commercial antiviral agent Ningnanmycin.
To confirm the potential inhibitory capacity of these compounds against TMV, on the basis of our previous bioassays, we further evaluated the EC50 of several target compounds against TMV.In Table 5, the antiviral curative activities of compounds 3c, 3f, and 3r, with corresponding EC50 values of 290.0, 234.0, and 373.6 μg/mL against TMV, were much better than that of ningnanmycin (386.2 μg/mL).The protective activities of 3e and 3r (EC50 of 324.8 and 291.1 μg/mL) against TMV were better than or near to that of ningnanmycin (297.1 μg/mL).When R1 was Ph (3c), 4-F-Ph (3f) and 3-CF3 (3r) groups, the corresponding compounds presented excellent curative activity against TMV.Moreover, good protection activity against TMV was observed when R1 was 4-F-Ph (3e) and 3-CF3-Ph (3r) groups.a The commercial antiviral agent Ningnanmycin.

Experimental
Instruments.solvents and reagents were purchased from Shanghai Titan Scientific Co., Ltd., were of analytical reagent grade or chemically pure, and were treated with standard methods prior to use.The reactions were monitored by thin-layer chromatography on silica gel GF254.Melting points (m.p.) of all synthesized compounds were determined when left untouched on an XT-4-MP apparatus from Beijing Tech.Instrument Co. (Beijing, China).Using tetramethylsilane (TMS) as the internal standard and chloroform as the solvent, 1 H, 13 C, and 31 P nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend-400 spectrometer (Bruker, Germany) and JEOL-ECX 500 NMR spectrometer (JEOL, Tokyo, Japan) operated at room temperature.High-resolution mass spectral (HRMS) data were performed with Thermo Scientific Q Exactive (Thermo). 3.1.Chemistry.

General Procedure for Preparation of Intermediates 2
To a stirred solution of (E)-4-(hydroxy-phenyl)-but-3-en-2-one (1.6 g, 0.01 mol) and aromatic aldehyde (0.011 mol) in ethanol (15 mL), a solution of NaOH was added in the form of 10% aqueous solution.The reaction mixture was stirred for 10 h at room temperature.Then, the mixture was diluted with a tenfold volume of water and neutralized with aqueous HCl.The resulting precipitate was separated and recrystallized from ethanol to obtain the yellow solid 1,5-diphenylpenta-1,4-diene-3-one 2.

General Procedure for Preparation of Title Compounds 3a−3r.
A solution of intermediate 2 (1.0 mmol) and Et3N (3.0 mmol) in CCl4 (30 mL) was stirred until dissolved.The mixture was stirred in an ice-water bath for 30 min, after adding the mixture of H-phosphonate (4.0 mmol) and CCl4 (6 mL) dropwise.The mixture was then removed from the ice-water bath and stirring continued at room temperature for 24 h.After the reaction was completed (as indicated by TLC), the solvent was removed under depressurization, and the residue was diluted with EtOAc (3×35 mL), and washed with 5% HCl (3×30 mL), and 5% NaOH (3×30 mL), respectively.the organic layer was dried by anhydrous Na2SO4, the solvent was removed under depressurization, and the residue was purified by column chromatography on silica gel to obtain the title compounds 3a-3r.Representative data for 3a are listed below.

Bioassays:Antibacterial Bioassays
The antibacterial effects of the target compounds against Xac (strain 29-1, Shanghai Jiao Tong University, China) and Xoo (strain PXO99A, Nangjing Agricultural University, China) were evaluated by the turbidimeter test [31][32][33].The title compounds were dissolved in 150 μL of dimethylformamide (DMSO) and diluted with water containing Tween-20 (0.1%) to obtain final concentrations of 100 and 50 μg/mL.DMSO in sterile distilled water served as blank control, and the commercial agent thiadiazole-copper was used as positive control.Approximately 1 mL of the sample liquid was added to a 15 mL tube, 4 mL of nutrient broth (NB, 1.5 g of beef extract, 0.5 g of yeast powder, 5.0 g of glucose, 2.5 g of peptone, and 500 mL of distilled water, pH 7.0 to 7.2) media.Then, approximately 40 μL of Xoo or Xac bacterium solution were added.The inoculated test tubes were incubated at 28 ± 1 °C and continuously shaken at 180 rpm for 24-48 h.The growth of the cultures was monitored with a spectrophotometer by measuring the optical density at 595 nm (OD595) given by corrected turbidity values.The relative inhibitory (I %) are calculated by the following formula, where Ctur represents the corrected turbidity value (OD595) of bacterial growth on untreated NB (blank control) and Ttur represents the corrected turbidity values (OD595) of bacterial growth on treated NB.

Purification of TMV
The upper leaves of N. tabacum cv.K326 were selected and inoculated with TMV, using previously reported methods for TMV purification [36].

Curative Activities of Compounds against TMV In Vivo
Using N. tabacum L. leaves of the same age as the test subjects, the TMV virus were dipped and inoculated on the whole leaves, which were scattered with silicon carbide beforehand [34,35].After about 60 min, the leaves were washed with water, and after drying, the compound solution was smeared onto the left side of leaf, and the solvent was smeared onto the right side.The local lesion numbers were counted after 3 to 4 days.Each compound was tested three times.The compound solution was smeared on the left side of leaf, while the solvent was smeared on the right side [34,35].The leaves were inoculated with virus 12 h later.Then, the leaves were washed with water after inoculation for 2 The local lesion numbers were counted after 3 to 4 days.Each compound was conducted three times.

Conclusions
In summary, with the aim to develop a novel, highly-efficient, and environmentally benign virucide, we introduced a H-phosphonate scaffold into penta-1,4-diene-3-one to synthesize 18 curcumin derivatives.Their antibacterial activities against Xac and Xoo in vitro and their antiviral activity against TMV in vivo were evaluated.Bioassay results showed that several of the title compounds exhibited good antibacterial and antiviral activities.Among these, compounds 3c, 3d, 3f, 3k, 3l, 3n, and 3o exhibited appreciable antibacterial activities against Xoo, with EC50 values of 22.9, 6.7, 43.5, 16.5, 11.4, 26.5, and 36.1 μg/mL, respectively.These were significantly better than those of commercial agents BT and TC (78.7 and 58.8 μg/mL).Compounds 3c, 3e, 3g, and 3o showed excellent antibacterial activities against Xac, with EC50 values of 10.6, 22.4, 18.4, and 10.8 μg/mL, respectively, which were obviously superior to those of commercial agents BT and TC (87.9 and 44.5 μg/mL).In addition, compounds 3c, 3f, and 3r showed remarkable curative activities against TMV, with EC50 values of 290.0, 234.0, and 373.6 μg/mL, respectively, which were better than that of ningnanmycin (386.2 μg/mL).Compound 3r exerted comparative protective activity against TMV, with an EC50 value of 291.1 μg/mL, which was better than that of ningnanmycin (297.1 μg/mL).Given the above results, these penta-1,4-diene-3-one derivatives, containing a H-phosphonate scaffold, should be further studied as potential alternative templates in the search for novel antibacterial and antiviral agents.

Table 2 .
EC50 values of the title compounds against Xoo in vivo .
a The commercial agricultural antibacterial agents Thiodiazole copper (TC) and Bismerthiazol (BT) were used as control agents.

Table 3 .
EC50 values of the title compounds against Xac in vivo.
a The commercial agricultural antibacterial agents Thiodiazole copper (TC) and Bismerthiazol (BT) were used as control agents.Preprints (www.

Table 4 .
Antiviral activities of the test compounds against TMV in vivo at 500 μg/mL.