Design, synthesis, and antibacterial activity of novel myricetin derivatives containing sulfonate

A series of myricetin derivatives containing sulfonate groups were designed and synthesized. Preliminary antibacterial activity showed that most of the target compounds exhibited significant biological activities against Xanthomonas axonopodis pv. Citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In particular, the EC50 value of compound 3e was 13.76 μg/cm3 against Xac, which was better than commercial reagents bismerthiazol (50.32 µg/cm3) and thiodiazole copper. (83.27 µg/cm3), and the EC50 value of compound 3j was 11.92 μg/cm3 against Xoo in vitro, The result was better than that of bismerthiazol (72.08 µg/cm3) and thiodiazole copper (99.26 µg/cm3). Compound 3j displayed the better in vivo activity against rice bacterial leaf blight than bismerthiazol and thiodiazole copper. Meanwhile, the antibacterial mechanism of compounds 3e and 3j was studied by scanning electron microscope (SEM). These results suggested that myricetin derivatives containing sulfonate can be considered as a new antibacterial reagents.


Introduction
Plant bacterial diseases are frequently encountered in agricultural production and difficult to manage, such as citrus canker, tobacco bacterial wilt and rice bacterial leaf blight, which can be caused by Xanthomonas axonopodis pv. Citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. oryzae (Xoo), respectively. Meanwhile, these bacterial diseases have a serious impact on the yield of crops every year [1][2][3]. Although there are many traditional agricultural fungicides on the market at present, the extensive application and abuse of antibacterial drugs have not only increased the resistance of pathogenic genes, but also caused serious environmental pollution and affect human health [4][5][6]. Therefore, it is of great significance to develop a new kind of antibacterial reagents with high efficiency and novel mechanism.
Sulfonate derivatives, owing to physicochemical properties, have a strong affinity for lipid phases and can cross the cuticle membrane easily to bind to target sites [23], and compounds containing aryl sulfonate moieties have received considerable attention due to extensive biological activities, and it has been proven to possess antiviral [24,25], antibacterial [26][27][28], insecticidal [29,30], anticancer [31,32], and other biological activity [33]. They were widely used in agricultural industries, medicine researches and other fields. We theorized that introducing sulfonate groups into myricetin might generate novel lead molecules with better physicochemical properties. Therefore, a series of novel myricetin derivatives containing sulfonate were designed (Fig. 2), synthesized (Scheme 1), and their in vitro and in vivo antibacterial activity was evaluated. To the best of our knowledge, this is the first report on the synthesis and

Results and discussion
The synthetic route of myricetin derivatives containing sulfonate is shown in Scheme 1. Myricetrin, methyl iodide, and potassium carbonate were mixed and stirred at room temperature to give intermediate 1 and then continued to react with concentrated hydrochloric acid to get intermediate 2. Intermediate 2 was treated with different benzenesulfonyl chlorides in the presence of potassium carbonate to obtain the target products 3a-3v in good yields ranging from 53 to 89%.
The structures of all compounds were confirmed by 1 H NMR, 13 C NMR, 19 F NMR, and HRMS. The representative data of compound 3b are shown below. In the 1 H NMR spectrum, the multiple signal δ = 6.57-7.57 ppm demonstrated the presence of aromatic ring protons, the singlet signal of 3.6-4.0 ppm belonged to -OCH 3 on myricetin, and the singlet signal of 2.34 ppm indicates the presence of -CH 3 group. Typical chemical shifts at δ = 145.45, 133.91, and 21.56 ppm in 13 C NMR spectrum confirmed the existence of -O-C-, -S-C-, and -CH 3 groups, respectively. In 19  To further confirm the structure of synthesized compounds, the molecular structure of 3n was studied as a representative example by single-crystal X-ray analysis. The tested single crystal was crystallized from the mixture of acetone and N,N-dimethylformamide solution under room temperature. The crystal diffraction data are presented in Table 1. Crystal structure diagram and crystal packing diagram are shown in Fig. 3A and B, respectively. Figure 3A shows that the intramolecular hydrogen bond C 4 ···O 1 , O 7 ···O 5 , O 5 ···C 8 in crystal combines with the skeleton of myricetin and sulfonate. As showed in Fig. 3B, the four intermolecular hydrogen bonds H 25 ···H 22 -C, H 18 A···O5, C-H 1 A···O 6 , and C-H 1 A···H 1 A-C constructed the three-dimensional structure of target compound 3n. The deposition number is CCDC-2017276.
In order to further confirm the antibacterial activities of title compounds, their EC 50 values were determined. The results indicated that compounds 3e, 3i, 3k, 3n, and 3o exhibited prominent antibacterial activity against Xac with EC 50 values of 13 19.53, and 11.92 μg/ cm 3 , respectively, which were better than BT (72.08 μg/cm 3 ) and TC (99.26 µg/cm 3 ). The results showed that these compounds could be further studied as potential compounds in search of novel antibacterial agents.
The control effect of compound 3j against rice bacterial leaf blight was determined by leaf-cutting method. As shown in Table 4 and Fig. 4, the effective curative activity against rice bacterial leaf blight was 40%, which was better than BT (35%) and TC (30%). Furthermore, 3j demonstrated better protection activity (47%) against rice bacterial leaf blight than BT (39%) and TC (27%). These results suggest that 3j effectively inhibit the growth of rice bacterial leaf blight under greenhouse conditions.

Scanning electron microscopy (SEM) studies
Through the analysis of antibacterial activity, the mechanism of 3e for Xac and 3j for Xoo were further studied via SEM. We found that the increase of concentration would lead to the deepening of cell membrane damage, in the control group without treatment with compound, the cell membrane were full and remains intact (Figs. 5A and 6D). Part of the cell membranes began to be destroyed when the concentration was 50 μg/cm 3 (Figs. 5B and 6E). And most of the cell membrane was destroyed when the concentration was increased to 100 μg/cm 3 , and only a few cells were remained unaffected (Figs. 5C and 6F). These results showed that the damage of cell membrane became more and more serious with the increasing of the compound concentration. These SEM images further confirmed that 3e and 3j destroyed the bacterial cell membrane and eventually killed the bacteria.

Conclusions
In conclusion, a series of myricetin derivatives containing sulfonate were designed and synthesized, the antibacterial activity of these derivatives against Xac, Rs, and Xoo have been tested in vitro, and the results indicated that most of compounds have good antibacterial activities. Especially compound 3e against Xac and compound 3j against Xoo wih the EC 50 values were 13.76, 11.92 µg/cm 3 , respectively, which were superior to BT (50.32 and 72.08 µg/cm 3 , respectively) and TC (83.2 and 99.2 µg/cm 3 , respectively). Compound 3j also displayed good antibacterial activities against rice bacterial leaf blight (curative activity was 40.7% and protective activity was 47.9%), which were superior to the curative and protection activities of BT (35% and 39%) and TC (30% and 27%). The SEM images of Xac treated with compound 3e and Xoo treated with compound 3j revealed that the cell membranes of bacteria are deformed and broken. These results demonstrate that novel myricetin derivatives containing sulfonate could be further studied as new antibacterial compounds.

Experimental
All reagents and solutions were purchased from Chemical Reagent Company and were analytical grade reagents. The melting point of all synthesized compounds were found by an XT-4 Binocular Microscope melting point apparatus (Beijing Tech. Instrument, China). DMSO-d 6 were used as a solvent and TMS as an internal standard, a Bruker Ascend-400 spectrometer (Bruker Optics, Switzerland) was used to give the 1 H NMR, 13 C NMR, and 19 F NMR spectra of title compounds. HRMS data were obtained using Thermo Scientific Q Exactive Hybrid Quadrupole Mass Spectrometer (Thermo Scientific Inc., St Louis, MO, USA). The X-ray crystal data were acquired using a Bruker D8-QUEST diffractometer (Bruker Optics, Switzerland). All 1 H NMR, 13 C NMR, and 19 F NMR spectra, and HRMS are provided in Supporting Information.

General synthesis procedure for intermediate 1 and intermediate 2
Based on the previously reported method [36][37][38], the synthetic route of target compound is listed in Scheme 1. The myricetin, anhydrous potassium carbonate and N,Ndimethylformamide (DMF) were added to a round-bottom flask with magnetic stirring; the reaction mixture was allowed to stir for 20 min at room temperature and potassium iodide was asses dropwise slowly. After completion of the reaction, monitored by TLC plate, the reaction mixture was extracted with dichloromethane and the solvent was removed under reduced pressure to obtain 5,7-dimethoxy-3-[(3,4,5-trimethoxy-6-methyltetrahydro-2H-pyran-2-yl)-  Intermediate 1 was added in ethanol and refluxed with stirring for 2 h. Concentrated hydrochloric acid was slowly added dropwise at this temperature, continued to reflux for 2-3 h, and a large amount of solid precipitated when the reaction was cooled to ambient temperature, which was filtered and dried to obtain 3-hydroxy-5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one (intermediate 2).

General synthesis procedure for target compounds 3a-3v
Intermediate 2, anhydrous potassium carbonate and acetonitrile were added to round-bottom flask with magnetic stirring and reflux for 0.5-1 h.
Then substituted benzenesulfonyl chloride was added and continued to reflux for 2-3 h until completion of the reaction as determined by TLC. The mixture was cooled to room temperature and poured into ice water; the crude products were recrystallized with anhydrous ethanol and DMF to give target compounds 3a-3v.            = 170.03, 164.92, 160.92, 158.75, 155.46, 153.02,  140.20, 139.35, 137.76, 133.30, 131.74, 130.03, 126. 13    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/.