Design, Synthesis, and Acaricidal Activity of Phenyl Methoxyacrylates Containing 2-Alkenylthiopyrimidine

A series of novel phenyl methoxyacrylate derivatives containing a 2-alkenylthiopyrimidine substructure were designed, synthesized, and evaluated in terms of acaricidal activity. The structures of the title compounds were identified by 1H NMR, 13C NMR and high-resolution mass spectra (HRMS). Compound (E)-methyl 2-(2-((2-(3,3-dichloroallylthio)-6-(trifluoromethyl)pyrimidin-4-yloxy)methyl)phenyl)-3-methoxyacr-ylate (4j) exhibited significant acaricidal activity against Tetranychus cinnabarinus (T. cinnabarinus) in greenhouse tests possessing nearly twice the larvicidal and ovicidal activity compared to fluacrypyrim. Furthermore, the results of the field trials demonstrated that compound 4j could effectively control Panonychuscitri with long-lasting persistence and rapid action. The toxicology data in terms of LD50 value confirmed that compound 4j has a relatively low acute toxicity to mammals, birds, and honeybees.


Introduction
Around 300,000 to 500,000 species of mites are distributed all over the world. Phytophagous mites are recognized as one of the most difficult pest communities to control, which can harm more than 150 crops, especially cotton, citrus, apples, vegetables, tea and flowers, causing great losses to agriculture and forestry every year [1][2][3][4][5][6][7][8]. On the other hand, due to the improper and frequent use of pesticides and a series of characteristics of mites themselves, such as the short generation cycle, rapid reproduction, high inbreeding rate, and high mutation rate, mites have already developed more and more serious resistance to the existing acaricides [9][10][11]. It is reported that 53 species of resistant ticks exist in the world [6]. Therefore, there is an urgent need for continually discovering and developing new acaricides with improved activity to safeguard crops against mites.
Compounds containing the methoxyacrylate moiety are widely applied as agrochemical acaricides [12]. Fluacrypyrim, the first strobilurin acaricide, was discovered by (Badische Anilin-und-Soda-Fabrik) BASF and developed by Nippon Soda [13]. It can effectively control all the growth stages of spider mites with a mode of action inhibiting mitochondrial electron transport in complex III within the respiratory chain [14]. Besides, Pyriminostrobin invented by the Liu group at Shenyang Sinochem Agrochemicals R&D Co., Ltd. and HNPC-A3066 discovered by Liu et al. at Hunan Research Institute of Chemical Industry, are another two typical strobilurin acaricides [15,16].
Molecules 2020, x, x 2 of 12 agricultural nematocides fluensulfone, 1,3-dicholorpropene; insecticides pyridalyl, flufiprole; fungicides fenpyrazamine, silthiofam and imazalil, and bactericides/fungicides/plant activators probenazole ( Figure 1) [17]. Furthermore, the synthesis of new pyrimidine derivatives bearing the alkenyl/haloalkenyl group has attracted much attention from many organic chemists. Substituted 2-(alkenyl/haloalkenyl)thio)-pyrimidine showed good herbicidal, fungicidal and insecticidal activity [18][19][20][21][22][23]. Organosulfur compounds are of fundamental and immense importance in organic chemistry. Sulfur-containing frameworks containing sulfur-containing heterocycles, sulfonamides, sulfonylureas, thioethers, sulfones, thioesters and thiocarbonyl compounds, have attracted considerable interest due to their unique physical and chemical properties [24][25][26][27], as well as their physiological activities [28][29][30]. Remarkably, in recent decades, thioether has emerged as the key functional groups widely used in natural products [31][32][33], medicine [24,25,34], pesticides [35,36], bioinformatics [37] and materials [28,38,39] (Figure 2). As a result, thioethers-containing molecules are attracting more and more attention based on their variety of biological activities [29]. Traditional synthetic procedures of fluacrypyrim involve a long synthesis step, low yield and poor cost performance. With an aim to find novel strobilurin acaricides with improved acaricidal activity and cost performance, and follow the isosteric design, we try to introduce the thioether group into strobilurin fluacrypyrim and construct a pyrimidine substructure bearing alkenyl/haloalkenyl at the same time. A series of novel strobilurin compounds were designed and synthesized ( Figure 3). The synthetic routes for the target compounds 4a-4u and for the intermediate 2a-2o, are displayed in Figure 4, and have the advantages of convenient synthesis, simple post-processing, and high yield. Traditional synthetic procedures of fluacrypyrim involve a long synthesis step, low yield and poor cost performance. With an aim to find novel strobilurin acaricides with improved acaricidal activity and cost performance, and follow the isosteric design, we try to introduce the thioether group into strobilurin fluacrypyrim and construct a pyrimidine substructure bearing alkenyl/haloalkenyl at the same time. A series of novel strobilurin compounds were designed and synthesized ( Figure 3). The synthetic routes for the target compounds 4a-4u and for the intermediate 2a-2o, are displayed in Figure 4, and have the advantages of convenient synthesis, simple post-processing, and high yield. The detailed synthesis, structure and activity relationship (SAR), and acute toxicology data are presented, which would provide the basis for further research.
Molecules 2020, x, x 3 of 12 The detailed synthesis, structure and activity relationship (SAR), and acute toxicology data are presented, which would provide the basis for further research.

Chemistry
The target compounds were characterized by 1 H NMR, 13 C NMR, melting point, high-resolution mass spectra (HRMS) analyses and some compounds were characterized by 19 F NMR analyses. All the spectral and analytical data were consistent with the assigned structures (Supplementary Materials).
It is well known that the homologous elements in the same group with a higher atomic number have a lower electronegativity, so they have the stronger electron-donating ability and thus greater nucleophilicity [40,41]. Therefore, under the same conditions, the reaction between the thiol at the 2position of the pyrimidine and halogenated alkenes could be well controlled by setting the reaction temperature between 40 and 50 °C, while the reaction between the halogenated alkenes and the hydroxyl at the 4-position of the pyrimidine could be reduced. The intermediate B is the key to synthesize the target compounds, and their synthesis procedures are shown in Figure 4. The physical properties of intermediates (1a-1f and 2a-2o) and target compounds (4a-4u) are presented in Table  1 and Table 2.

Chemistry
The target compounds were characterized by 1 H NMR, 13 C NMR, melting point, high-resolution mass spectra (HRMS) analyses and some compounds were characterized by 19 F NMR analyses. All the spectral and analytical data were consistent with the assigned structures (Supplementary Materials).
It is well known that the homologous elements in the same group with a higher atomic number have a lower electronegativity, so they have the stronger electron-donating ability and thus greater nucleophilicity [40,41]. Therefore, under the same conditions, the reaction between the thiol at the 2position of the pyrimidine and halogenated alkenes could be well controlled by setting the reaction temperature between 40 and 50 °C, while the reaction between the halogenated alkenes and the hydroxyl at the 4-position of the pyrimidine could be reduced. The intermediate B is the key to synthesize the target compounds, and their synthesis procedures are shown in Figure 4. The physical properties of intermediates (1a-1f and 2a-2o) and target compounds (4a-4u) are presented in Table  1 and Table 2.

Chemistry
The target compounds were characterized by 1 H NMR, 13 C NMR, melting point, high-resolution mass spectra (HRMS) analyses and some compounds were characterized by 19 F NMR analyses. All the spectral and analytical data were consistent with the assigned structures (Supplementary Materials).
It is well known that the homologous elements in the same group with a higher atomic number have a lower electronegativity, so they have the stronger electron-donating ability and thus greater nucleophilicity [40,41]. Therefore, under the same conditions, the reaction between the thiol at the 2-position of the pyrimidine and halogenated alkenes could be well controlled by setting the reaction temperature between 40 and 50 • C, while the reaction between the halogenated alkenes and the hydroxyl at the 4-position of the pyrimidine could be reduced. The intermediate B is the key to synthesize the target compounds, and their synthesis procedures are shown in Figure 4. The physical properties of intermediates (1a-1f and 2a-2o) and target compounds (4a-4u) are presented in Tables 1  and 2.

Acaricidal Activity
The acaricidal activity results of all the title compounds to control adult Tetranychus cinnabarinus (T. cinnabarinus) by using the spraying method in the greenhouse are listed in Table 3. Table 3. Acaricidal activities of the compounds 4a-4u against the adults of T. cinnabarinus.

Activities (%) against Adults at
a refers to not tested.
continuously in the following optimization.
Finally, based on the valuable SARs information achieved above, the R2 of 3,3-dichloroallyl and methoxyacrylate, we went back to modify the R1 of the pyrimidine ring with opposite electron effect groups like electron-donating methyl, ethyl, n-Pr and cyc-Pr. Unfortunately, all of the four compounds 4r-4u did not display any activity, even at 500 mg L −1 .
Finally, based on the valuable SARs information achieved above, the R 2 of 3,3-dichloroallyl and methoxyacrylate, we went back to modify the R 1 of the pyrimidine ring with opposite electron effect groups like electron-donating methyl, ethyl, n-Pr and cyc-Pr. Unfortunately, all of the four compounds 4r-4u did not display any activity, even at 500 mg L −1 .

Field Trials
In this study, the compound 4j was selected as the most potent compound with the highest acaricidal activity against T. cinnabarinus. Field trials were carried out to confirm its field performance in November, 2018 in Nanning, Guangxi Province, China. The field trials (Table 5) indicated that the acaricidal activity of compound 4j at 100 mg L −1 against citrus red mites was roughly equivalent to that of cyenopyrafen and slightly higher than that of cyetpyrafen, a newly commercialized acaricide from Sinochem Group. At a concentration of 100 mg L −1 , the effect of compound 4j on controlling the citrus red mite was 96.61, 97.36, 96.25, and 81.02% after 3, 7, 10, and 20 days of treatment, respectively, while cyenopyrafen revealed a controlling effect of 92.26, 97.85, 96.43, and 80.87% against Panonychuscitri after 3, 7, 10, and 20 days of treatment, respectively. At the same concentration, the effect of cyetpyrafenon controlling citrus red mite was also 97.70, 100, 99.38, and 89.79% after 3, 7, 10 and 20 days of treatment, respectively. The comparison of the above-treatment results indicated that the compound 4j was a highly active and rapid-acting acaricidal compound, which can effectively control Panonychuscitri at an application rate of 100 mg L −1 . Table 5. Acaricidal activity of the compound 4j against P. Citri in field tests (Nanning, China). The significance level is 0.05.

The Toxicity of Compound 4j
The toxicity of compound 4j on primary mammals, birds, and honeybees was evaluated by The Safety Evaluation Center of Shenyang Research Institute of Chemical Industry (National Shenyang New Drug Safety Evaluation and Research Center, Shenyang, China) and Zhejiang Academy of Agricultural Science in 2019 as presented in Table 6. It was found that (1) the acute oral median lethal dose (LD 50 ) of compound 4j for rats is higher than 500 mg kg −1 ; (2) it has a low toxicity for birds (>2090 mg a.i./kg) and honeybees (>116 µg a.i./honeybee); and (3) it moderately irritates rabbits' eyes.

Reagents and Instruments
The reagents were all high purity analytical or chemical grades, purchased from commercial sources and were used as received. All anhydrous solvents were dried and distilled by standard techniques before use. The melting points were determined by using an RY-1 melting point apparatus (TaiKe, Beijing, China). The 1 H NMR, 13 C NMR, and 19 F NMR spectra were recorded utilizing an AV 400/500 spectrometer (Bruker, Karlsruhe, Germany) in CDCl 3 or DMSO-d 6 solution using tetramethylsilane as an internal standard, and the chemical shifts (δ) were given in parts per million (ppm). High-resolution mass spectra (HRMS) data were obtained by employing Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) (ionspec, 7.0 T). The reaction progress was monitored by thin-layer chromatography (TLC) on silica gel GF 254 (Tianzhe, Qingdao, China), and the spots were visualized with ultraviolet (UV) light (Gonyi, Zhengzhou, China).

Synthetic Chemistry
All the title compounds were synthesized as described in Figure 4 (Page 6). General procedures for the preparation of the title compounds (4a-4u) and intermediates (1a-1f and 2a-2o) according to the related literature [44][45][46] and spectra data of the title compounds (4a-4u) are provided in the Supplementary Materials. Intermediate 3a was obtained from 1H-isochromene-3(4H)-one as the starting material via a multi-step synthesis referring to the literatures [47,48]. Intermediate 3b was synthesized using a 2-methylbenzoic acid as the starting material [49,50].

Synthesis of 6-Substituent
All of the thiouracil derivatives (R 1 = CH 3 , C 2 H 5 , n-Pr, cyc-Pr, CHF 2 , CF 3 ) were prepared as followed. To a 1000 mL three-neck flask equipped with a magnetic stirrer, a thermometer, and a dropping funnel, 500 mL of methanol and 59.4 g (1.1 mol) of solid sodium methoxide were added, and the mixture was then stirred at room temperature. After 0. Potassium carbonate (15.5 g, 98.0%, 0.11 mol) was added into a solution of 14.5 g (0.10 mol) of 6-trifluoromethyl-2-thiouracil (1b, R 1 = CF 3 ) in 100 mL of DMF, and the reaction mixture was stirred at 40 • C for 0.5 h, followed by the addition of 19.1 g (0.10 mol) of 4-bromo-1,1,2-trifluorobut-1-ene; then, the reaction mixture was heated to 50 • C for about 3 h and monitored by TLC until the reaction was complete; afterward, the mixture was poured into water, and a 2 M HCl aqueous solution was added to the flask until the reaction mixture was neutralized. The resultant solid was separated by filtration, then washed with water, and finally dried in a desiccator to obtain 28.0 g of the intermediate 2f as a white solid at a yield of 92%; melting point: 82.6-83.7 • C; 1 H NMR (500 MHz, DMSO-d 6 ) δ13.33 (br, 1H, OH), 6.55 (s,1H, pyrimidy1-H), 3.26 (t, J = 9.0 Hz, 2H, CH 2 ), 2.69-2.75 (m, 2H, CH 2 ).

Acaricidal Activities Assay
The detailed procedure of the acaricidal activity against adult T. cinnabarinus was measured according to the related literature [51].
Each of the test compounds was first dissolved in a mixture of acetone and water (at a volume ratio of 9 to 1) containing 0.1% TWEEN ® 80 to furnish the stock solutions. A series of test solutions were then prepared by diluting the stock solutions with water containing 0.1% TWEEN ® 80. Afterward, the kidney bean plants with one true leaf were infested with T. cinnabarinus (carmine spider mite) prior to spraying. An airbrush was used to spray the plants with the test solutions, and each bioassay was replicated three times at a temperature of 25 ± 1 • C to meet the statistical requirements. After the plants were dried, they were transferred to a maintenance room for observation. The mortality rate of the spider mite was investigated after 72 h. The mites that did not either fly away or respond to the touch of a fine brush were considered to be dead. The rate of mortality was evaluated according to a percentage scale of 0-100, in which 0 indicates no activity of the test solutions against the spider mites, and 100 indicates totally killing them. When the percentage of the mortality of the blank control was less than 5%, the results of the treatment were directly used. However, if the percentage of the mortality of the blank control ranged from 5 to 20%, the results were corrected by the means of the following equation: where, V is the value of the corrected percentage of mortality, and X represents the viability of the blank control; Y stands for the viability of the treatment. As a positive control, commercial fluacrypyrim was also evaluated using exactly the same procedure.

Conclusions
In summary, a series of phenyl methoxyacrylates containing 2-alkenylthiopyrimidine derivatives were designed, synthesized, and evaluated in terms of acaricidal activity. Compound 4j exhibited significant acaricidal activity against the adults, larvae, and eggs of T. cinnabarinus (Boisduval), significantly higher than fluacrypyrim. Furthermore, the field trials proved that the acaricidal efficacy of compound 4j was approximately equivalent to those of cyetpyrafen and cyenopyrafen. The field trials also demonstrated that compound 4j at a concentration of 100 mg L −1 presented a control effect of 96.25% and 81.02% against Panonychuscitri after 10 and 20 days of treatment, respectively, indicating that it has long-lasting persistence (20 days) in field trials. It also acts rapidly against Panonychuscitri by reaching a controlling effect of 96.61% after 3 days of treatment. The compound 4j has quite low acute toxicity to mammals, birds, and honeybees. Finally, the findings of the current work suggested that the compound 4j (3,3-dichloroallylthio, trifluoromethyl, pyrimidine) could be a novel acaricide candidate for the control of spider mites, which is worthy of being further studied.