Design, Synthesis and Antibacterial Activity of Novel Pyrimidine‐Containing 4H‐Chromen‐4‐One Derivatives **

A series of pyrimidine‐containing 4H‐chromen‐4‐one derivatives were designed and synthesized by combining bioactive substructures. Preliminary biological activity results showed that most of the compounds displayed significant inhibitory activities in vitro against Xanthomonas axonopodis pv. Citri (X. axonopodis), Xanthomonas oryzae pv. oryzae (X. oryzae) and Ralstonia solanacearum (R. solanacearum). In particular, compound 2‐[(3‐{[5,7‐dimethoxy‐4‐oxo‐2‐(3,4,5‐trimethoxyphenyl)‐4H‐1‐benzopyran‐3‐yl]oxy}propyl)sulfanyl]‐4‐(4‐methylphenyl)‐6‐oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (4c) demonstrated a good inhibitory effect against X. axonopodis and X. oryzae, with the half‐maximal effective concentration (EC50) values of 15.5 and 14.9 μg/mL, respectively, and compound 2‐[(3‐{[5,7‐Dimethoxy‐4‐oxo‐2‐(3,4,5‐trimethoxyphenyl)‐4H‐1‐benzopyran‐3‐yl]oxy}propyl)sulfanyl]‐4‐(3‐fluorophenyl)‐6‐oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (4h) showed the best antibacterial activity against R. solanacearum with an EC50 value of 14.7 μg/mL. These results were better than commercial reagents bismerthiazol (BT, 51.7, 70.1 and 52.7 μg/mL, respectively) and thiodiazole copper (TC, 77.9, 95.8 and 72.1 μg/mL, respectively). In vivo antibacterial activity results indicated that compound 4c displayed better curative (42.4 %) and protective (49.2 %) activities for rice bacterial leaf blight than BT (35.2, 39.1 %) and TC (30.8, 27.3 %). The mechanism of compound 4c against X. oryzae was analyzed through scanning electron microscopy (SEM). These results indicated that pyrimidine‐containing 4H‐chromen‐4‐one derivatives have important value in the research of new agrochemicals.


Introduction
Plant diseases can cause huge losses in the yield of economic crops every year, which one of the main reasons is bacterial infection. Such as, the citrus canker, tobacco bacterial wilt and rice bacterial leaf blight are caused by Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae, respectively. [1 -3] These bacteria are very serious and hard to manage effectively in agricultural production. Therefore, these agrochemicals (bismerthiazol, thiodiazole-copper and azoxy strobin) are often used to prevent bacterial infection. [4,5] But prolonged and abuse of traditional chemical pesticides will enhance the resistance of the bacterial, and are harmful to the environment and human health. [6][7][8] Thus, to explore novel antibacterial agents with highly-efficient, broadspectrum, and environmentally is still an arduous task in pesticide science.
3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphen-yl)-4Hchromen-4-one (called "myricetin") belongs to a class of natural products which are widely found in fruits, vegetables, and herbs. It has attracted the attention of more and more researchers, due to the unique chemical structure and diverse biological activities. [9] Pharmacological research have shown that myricetin and its derivatives present antiviral, [10,11] antibacterial, [12 -14] antioxidation, [15,16] anticancer, [17 -19] antiinflammatory [20] and other biological properties, but most of them are limited to the field of medicine and have few applications in agriculture. In previous work, myricetin was took as the lead compound and active fragments were introduced, a mass of myricetin derivatives with significant biological activity were discovered. In 2017, Zhong described myricetin derivatives containing 1,3,4thiadiazole structure, which displayed good inhibitory effect on phytopathogenic bacteria. [21] Recently, Jiang reported a series of dithiocarbamatecontaining 4H-chromen-4-one derivatives, which have good in vitro antibacterial activity against X. axonopodis and X. oryzae, with an EC 50 values were 0.11 and 1.58 μg/mL, respectively, which were far better than commercial agents bismerthiazol and thiodiazole copper. [22] Pyrimidine is an important class of heterocyclic compounds, which has a wide range of applications in medicine and pesticides research, due to its remarkable biological and pharmacological properties, such as antibacterial, [23 -25] antiviral, [26,27] insecticide, [28,29] anticancer, [30,31] antimalarial, [32] herbicida [33] and other biological activities. [34] In 2019, Zhang reported a series of derivatives containing pyrimidine ethers were synthesized, which have a good control effect on cucumber downy mildew. [35] Recently, Bai described that some pyrimidine derivatives have significantly inhibitory efficiencies against Gram-positive bacteria, Gram-negative bacteria and the fungus Candida albicans. [36] In order to develop novel, highly efficient and environment friendly bactericides, we introduced the pyrimidine moiety into 4H-chromen-4-one, and a variety of pyrimidine-containing 4H-chromen-4-one derivatives were synthesized and their antibacterial activity were evaluated (Figure 1).

Chemistry
The structures of the title compounds 4a-4x were characterized by 1 H-NMR, 13 C-NMR, 19 F NMR and HRMS. In 1 H-NMR spectra, multiplet signals at δ 8.00 -6.50 ppm shown the presence of protons in aromatic nucleuses, and a singlet at δ 4.00 -3.70 ppm revealed the presence of -OCH 3 group, the -O-CH 2 -and -S-CH 2characteristic groups between the 4H-chromen-4-one skeleton and the substituted pyrimidine were observed at 4.10 -3.37 ppm. The chemical shifts of 13 C-NMR spectra were 172.57 -160.77 ppm, 109.12 -106.14 ppm, 27.80 -32.12 ppm in which confirmed the existence of C=O, -CN and -CH 2 -groups, respectively. The HRMS spectra of the title compounds shown absorption signals of [M + H] + ions that was consistent with their molecular weight.
To further confirm the structures of the title compounds, compound 4g was successfully cultured a single crystal and studied by single-crystal X-ray analysis as a representative example. The tested single crystal was crystallized from DMSO and acetone mixture solution containing compound 4g. As shown in Figure 2B, C00T…O007 and O00A…C01A were two important intramolecular hydrogen bonds, which combine with 4H-chromen-4-one and pyrimidine heterocycle fragments to construct the main molecular scaffold of target compound 4g. Besides, as presented in Figure 2C, four intermolecular hydrogen bonds (O008…H01B, C00J…C00L, C00J…C00L, H01B…O008) between neighboring molecules were primary forces for establishing the three-dimensional structure of target compound 4g. crystallographic data listed in Table 1. The deposition number is CCDC 2022970.

Antibacterial Activity of the Title Compounds against X. axonopodis, X. oryzae and R. solanacearum in Vitro
The in vitro antibacterial activities of the title compounds 4a-4x against X. axonopodis, X. oryzae and R. solanacearum were determined using turbidimeter tests, and the bismerthiazol, thiadiazole copper, and myricetin were tested as the controls. Preliminary bioassays results in Table 2 indicated that some compounds with good antibacterial activities against X. axonopodis, X. oryzae and R. solanacearum. The inhibition rates of the compounds 4b, 4c, 4h and 4o against X. axonopodis at 100 and 50 μg/mL were 80. Based on the preliminary bioassays results in Table 2, the EC 50 values of some target compounds against X. axonopodis, X. oryzae and R. solanacearum were further evaluated as shown in Table 3. Compounds 4c, 4h and 4o exhibited fine antibacterial activities against X. axonopodis, with the EC 50 values of 15.5, 28.3, and 29.4 μg/mL, respectively, which exceeded BT (50.3 μg/mL), and TC (83.2 μg/mL). Compounds 4c, 4h, 4n and 4p demonstrated good inhibitory effects against X. oryzae with the EC 50 values of 14.9, 23.8, 28.6 and 24.8 μg/mL, which were better than BT (72.0 μg/mL) and TC (99.2 μg/mL). Compounds 4c, 4d, 4h, 4k, 4l and 4o have better antibacterial activity against R. solanacearum with the

Structure-Activity Relationships of Antibacterial Activities
As indicated in Tables 2 and Table 3, the R groups had significant impact on the antibacterial activity, for example,

In Vivo Bioactivity of 4c Against Rice Bacterial Leaf Blight
The antibacterial test shown that compound 4c has significant anti-X. oryzae biological activity in vitro. To further verify the antibacterial activity of 4c, a pot experiment was carried out in vivo, and the experiments of 4c against rice bacterial leaf blight were determined using a leaf-cutting method. As shown in Table 4 and Figure 3, when the concentration of

Scanning Electron Microscopy (SEM) Study
The mechanism of compound against X. oryzae was analyzed by SEM. As shown in Figure 4, the surface morphology of X. oryzae were smooth and remains intact without compound to treatment ( Figure 4A). When the bacteria were treated with compound 4c at concentration of 50 μg/mL, bacteria cell membrane began to appear different degrees wrinkled ( Figure 4B). [a] Average of three replicates. [c] The commercial agricultural antibacterial agents bismerthiazol (BT) and thiodiazole-copper (TC) were used as control agents. When the concentration of compound increased to 100 μg/mL, the bacterial cell membrane showed invagination and rupture ( Figure 4C). Therefore, the compound 4c destroyed the cell membrane structure, to achieve the purpose of antibacterial.

Conclusions
In summary, aiming to develop novel and highefficient agents with better biological activities, a series of pyrimidine-containing 4H-chromen-4-one derivatives were designed and synthesized. In vitro  antibacterial bioassays showed that most of compounds exhibited significant antibacterial activities against X. axonopodis, X. oryzae and R. solanacearum.
In particular, compound 4c exhibited the best antibacterial activities against X. axonopodis, X. Oryzae, and compound 4h exhibited the better antibacterial activity against R. solanacearum than TC and BT. In vivo antibacterial bioassays shown that compound 4c was more effective than BT and TC in reducing bacterial blight of rice. Furthermore, the Scanning electron microscopy of 4c against X. oryzae showed that compound destroyed the cell membrane structure of the bacteria, and the damage of the cell membrane was more serious as the concentration increased. These results indicated that the pyrimidinecontaining 4H-chromen-4-one derivatives have better antibacterial activity and could be further study as potential replacement templates in the search for novel antibacterial agents.

Experimental Section
General All solvents and reagents were purchased from Shanghai Titan Scientific Co., Ltd, and were analytical grade or chemically pure. The melting point of all compounds (4a -4x) were determined by the X-4B melting point apparatus (Beijing Tech Instrument Co, Beijing. China). 1 H-NMR, 13 C-NMR and 19 F NMR were obtained on Bruker Ascend-400 spectrometer (Bruker Optics, Germany) Tetramethylsilane (TMS) as internal standard and DMSO was used as solvent; Mass spectral studies were performed on a quadrupole electrostatic field orbitrap mass spectrograph (Thermo Scientific, USA). The synthetic route of pyrimidine-containing 4H-Chromen-4-one derivatives was shown in Scheme 1.

Chemistry
General Procedure for the Synthesis of Intermediate 1 Based on the previous reported method, [26,37] the ethyl cyanoacetate (5 mmol), substituted aromatic aldehyde (5 mmol), thiourea (5 mmol) and anhydrous K 2 CO 3 (7.5 mmol) were added in ethanol (50 mL) for refluxed for about 4 -6 h. After the reaction was completed (the reaction was monitored by TLC), mixture was cooled to room temperature and diluted with ice water, a large amount of solids was precipitated as the pH adjusted with glacial acetic acid to weak acidity, the residue was filtered and dried with suction, recrystallized with absolute ethanol to obtain Intermediate 1 General Procedure for the Synthesis of Intermediate 2 Myricetin iodomethane and anhydrous K 2 CO 3 were stirred in N, N-dimethylformamide (DMF) for 48 h, and then, it was extracted with methylene chloride and concentrated. Then deglycosylation with concentrated hydrochloric acid under reflux in ethanol to prepare 3hydroxy-3',4',5',5,7-pentamethoxymyricetin (Intermediate 2). [22] General Procedure for the Synthesis of Intermediate 3 Intermediate 2 (2 mmol) and K 2 CO 3 (3 mmol) were dissolved in certain amount of N, N-dimethylformamide (DMF), stirred at room temperature for 0.5 -1 h, the different dibromoalkane (2 mmol) was added and the reaction system was stirred at room temperature for 10 h. Mixture was dispersed in 50 mL ice water when the reaction completed(the reaction was monitored by TLC), a white solid separated out, the liquid was removed by filtration, and the solid was continuously stirred in a mixed solution of 30 mL petroleum ether and ethyl acetate for 3-4 h, then, filtered and dried under reduced pressure to obtain Intermediate 3. [38] General Procedure for the Synthesis of Title Compounds 4a-4x A solution of intermediates 1 (3 mmol), intermediates 3 (3 mmol) and K 2 CO 3 (4.5 mmol) in 50 mL DMF was stirred under reflux for 4 -5 h, after reaction was completed (the reaction was monitored by TLC). The mixture was cooled to room temperature, then, poured into 80 mL ice water and extracted with ethyl acetate (3 × 30 mL), organic layer was washed with 5 % hydrochloric acid solution, saturated NaHCO 3 aqueous solution, saturated NaCl solution successively and dried with anhydrous Na 2 SO 4 . Solvent removed under vacuum and crude product was separated by column chromatography with petroleum ether/ethyl acetate (V : V = 1 : 1) to obtain the title compound 4a. Based on the similar method, the title compounds 4b-4x were prepared. AII the Spectroscopic data and Spectrographic of compound 4a-4x were provided in Supporting Material.

Bacteriostatic Activity Test in Vitro
The in vitro antibacterial activities of all the target compounds against X. axonopodis, X. oryzae and R. solanacearum were determined by turbidimetric method. [39] The test method was provided in Supporting Information.
In Vivo Antibacterial Activity The protection and curative activities of compound 4c against bacterial blight of rice were tested through potted plants using complete randomized block design according to a previous method with slight modifications. [38] Commercial agents BT and TC were used as positive control. The test method was provided in Supporting Information.

Scanning Electron Microscopy
To explore the mechanism of compounds 4c against X. oryzae, scanning electron microscopy (SEM) analysis was taken according to previous method. [40] This method is provided in Supporting Information.