Methoxyacrylate-based fungicide and methods for preparing and using the same

Preparation of Compound of Formula (I)

(E)-methyl 3-methoxy-2-(2-((5-methoxy-benzothiazol-2-ylthio)methyl)phenyl)acrylate

Preparation of Intermediate (II)

244 g of sodium polysulfide solution was mixed with 30 g of 1-chloro-4-methoxy-2-nitrobenzene, and then 20 mL of carbon disulfide was added with stiffing. The mixture was heated and refluxed for about 5 hrs and a yellow solid precipitated. The mixture was filtered and the filtrate was diluted with water to 1000 mL. An orange yellow solution was obtained. The solution was neutralized with hydrochloric acid (6M) to yield a light yellow solid. The solid was filtered, washed with water, and dried. 29.4 g of products was obtained with a yield of 93.1%.

Preparation of Intermediate (2): methyl 2-o-tolylacetate

To a flask (1000 mL), 120 g of 2-methylphenylacetic acid and 400 mL of anhydrous methanol were added, and then 24 mL of concentrated sulfuric acid was further added with stiffing. The mixture was heated with an oil bath and refluxed. The reaction was monitored with thin layer chromatography (TLC) until the reactants were basically consumed. Subsequently, the solvent was removed by vacuum distillation. The residue was dumped into 400 mL of water and extracted with 400 mL of ethyl acetate. The organic layer was washed with water (2×200 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. A light yellow oily product was obtained. The light yellow oily product was further distilled under vacuum condition to yield 117.3 g of a colorless oily product (135-140° C., 16 mmHg) with a yield of 92%.

Preparation of Intermediate (3): methyl 3-hydroxy-2-o-tolylacrylate

To a three neck flask (1000 mL), 50 g of methyl 2-o-tolylacetate and 600 mg of toluene were added, and then 54.3 g of sodium methoxide was further added with stiffing. The mixture was cooled with an ice-water bath to less than 10° C., and then a mixture of methyl formate (65 mL) and toluene (50 mL) was dripped. After that, the ice-water bath was removed and the temperature was raised slowly to room temperature. The solution was allowed to react with stirring for 12 hrs, and then dumped into 1000 mL of water, shaken, standed for demix. The organic layer was extracted with 500 mL of water twice. The water layer was combined, acidized with 6M hydrochloric acid to pH value of 4-5, extracted with ethyl acetate, dried with anhydrous magnesium sulfate, and distilled under vacuum condition to yield 55.9 g of light red oily product with a yield of 95%.

Preparation of Intermediate (4): methyl 3-methoxy-2-o-tolylacrylate

To a flask (1000 mL), 48 g of methyl 3-hydroxy-2-o-tolylacrylate, 54.4 g of potassium carbonate, and 500 mL of ethylene glycol dimethyl ether were added. The mixture was stirred at room temperature for an hour, and then 36 mL of dimethyl sulfate was added. The mixture was allowed to react for between 6 and 8 hrs with stiffing at room temperature. The reaction was monitored with thin layer chromatography (TLC) until the reactants were basically consumed. Subsequently, the mixture was filtered, the solvent was removed by vacuum distillation, the residue was washed with water (1000 mL), extracted with ethyl acetate (2×300 mL), dried with anhydrous sodium sulfate and concentrated under reduced pressure to yield a light yellow oily product with a yield of 97%.

Preparation of Intermediate (III)

39.14 g of methyl 3-methoxy-2-o-tolylacrylate, 41.3 g of N-bromosuccinimide (NBS), and 1.44 g of azobisisobutyronitrile (AIBN) were mixed with 200 mL of carbon tetrachloride. The mixture was lighted with an incandescent lamp for 6 hrs, cooled, and filtered. The filtrated was concentrated under reduced pressure to yield a yellow viscous material which could be used directly for further reaction.

Preparation of the Compound of Formula (I)

To 350 mL of an aqueous solution having 8.0 g of sodium hydroxide, 31.8 g of Intermediate (II) was added and stirred for 20 min. Subsequently, 70 mL of N, N-dimethylformamide (DMF) solution having 54 g of Intermediate (III) was added. The mixture was stirred at room temperature for 6 hrs and water added until the total volume was 1000 mL. The solution was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the product purified by chromatography over silica gel eluted with ethyl acetate/hexane, 52.1 g of a yellow solid was obtained with a total yield was 80.5%. m.p. 85-87° C. 1 H NMR (400 MHz, CDCl 3 ) δ: 3.72 (s, 3H, COOCH 3 ), 3.82 (s, 3H, ═CH—OCH 3 ), 3.84 (s, 3H, OCH 3 ), 4.52 (s, 1H, CH 2 ), 6.94 (dd, J=2.4 Hz, J=8.8 Hz, 1H, ArH), 7.16 (t, J=4.6 Hz, 1H, ArH), 7.29-7.31 (m, 2H, ArH), 7.40 (d, J=2.8 Hz, 1H, ArH), 7.54-7.61 (m, 3H, ═CH—OCH 3 , ArH). EI MS: m/z (%) 401 (M+, 4), 369(19), 205 (11), 167 (24), 144 (100), 101 (37). Anal. Calcd for C 20 H 19 NO 4 S 2 : C, 59.83; H, 4.77; N, 3.49. Found: C, 59.89; H, 5.01; N, 3.27.

FIG. 1 shows the crystal structure of the compound of Formula (I).

Evaluation on Indoor Bioactivity of Compound of Formula (I)

To verify the bioactivity of the compound of Formula (I), experiments evaluating indoor bioactivity comprising fungicidal activity test on potted plants at room temperature, fungicidal spectrum assay (in vitro), and in vivo micro-screening assay are carried out.

1. Fungicidal Activity Assay on Potted Plants at Room Temperature

Take cucumber downy mildew and powdery mildew as examples, the assay were carried out as follows:

1) Pseudoperonospora cubensis : Pathogen of Cucumber Downy Mildew

Two cucumber seedlings (the growing point had been removed) having the same growth status and in the true leaf stage were collected, sprayed with the compound of Formula (I), and dried naturally for 24 hrs for inoculation. Fresh cucumber leaves infected by downy mildew were collected, and sporangium on the back was rinsed with distilled water (about 10° C.) dipped with a brush. The obtained sporangium was prepared into a suspension (2-3×10 5 cells/mL). The suspension was sprayed uniformly with an inoculation sprayer (0.1 MPa) to the naturally-dried cucumber seedlings. Subsequently, the seedlings were cultured in an artificial climate chamber. The relative humidity was maintained at 100%, with temperature of between 15 and 20° C. 24 hrs later, the relative humidity was maintained at 90%, with temperature of between 15 and 24° C. 5 days later, compared with the control, the disease of the cucumber seedlings was graded. Based on the resultant disease index, the efficacy was calculated.

2) Sphaerotheca uliginea : Pathogen of Cucumber Powdery Mildew

A cucumber seedling having uniform growth status and in the true leaf stage was collected, sprayed with the compound of Formula (I), and dried in the shade for 24 hrs. Fresh spore was collected from cucumber leaves infected by Sphaerotheca uliginea , filtered with a double-layer gauze, and prepared into a suspension (1.0×10 5 cells/mL). The suspension was sprayed to the dried cucumber seedling. Subsequently, the inoculated seedling was dried naturally and cultured under light in a thermostatic chamber (21-23° C.). 7-8 days later, compared with the blank control, the disease of the cucumber seedlings was graded. Based on the resultant disease index, the efficacy was calculated.

3) Calculation Method

The grading standard follows “Guidelines on efficacy evaluation of pesticides”, and the efficacy is calculated on the basis of the disease index.

Disease index=Σ(total amount of diseased leaves of a grade×value of the grade)×100/(total amount of leaves×9);

Efficacy (%)=(disease index of blank control−disease index of experiment group)×100/disease index of blank control

The results are listed in Table 1 and Table 2.

2. Fungicidal Spectrum Assay (In Vitro)

To broaden the fungicidal spectrum of the compound of Formula (I), as targets the following 20 pathogens were assayed in vitro. The results showed the compound of Formula (I) had a wide fungicidal spectrum, particularly effective on Helminthosporum maydis, Ustilaginoidea virens , and Diaporthe medusa in vitro. The 20 pathogens and resultant plant diseases were:

Rice sheath blight ( Rhizoctonia solani ), Cucumber gray mold ( Botrytis cinerea ), Rape sclerotinia rot ( Sclerotinia sclerotirum ), Wheat scab ( Gibberella zea ), Rice blast ( Pyricularia oryzae ), Cucumber anthracnose ( Colletotrichum lagenarium ), Potato late blight ( Phytophthora infestans ), Pepper phytophthora blight ( Phytophthora capsici ), Southern leaf blight of corn ( Helminthosporum maydis ), Tomato early blight ( Alternaria solani ), Apple ring rot ( Botryosphaeria berengeriana ), Tobacco brown spot ( Alternaria alternata ), Banana leaf spot ( Helminthosporium torulosum ), Wheat sheath blight ( Rhizoctonia solani ), Wheat root rot ( Bipolaris sorokiniana ), Cotton wilt ( Fusarium oxysporum ), Citrus stem-end rot ( Diaporthe medusae ), Cotton boll rot ( Fusarium moniliforme ), Rice false smut ( Ustilaginoidea virens ), and Peanut brown spot ( Cercospora arachidicola ).

Measurement of In Vitro Inhibitory Rate

5 wt % formulation of the active compound of Formula (I) was prepared into a solution (300 mg/L). To a sterilized Erlenmeyer flask, 2 mL of the solution and 18 mL of PDA culture medium (50° C.) were added. The mixture was shaken uniformly and dumped into two dishes whose diameter was 9 cm to yield two toxic PDA culture mediums having a concentration of 30 mg/L. Various cultured pathogens were punched at the edge of colony by a puncher having a diameter of 5 mm respectively, and the obtained pieces were transferred to the center of the toxic PDA culture medium by an inoculating needle and cultured in an incubator at 25° C. Repeat the above steps. According to the growth status of CK colony, the colony diameter of pathogens was measured by crossing method with a caliper and thereby a corrected inhibition percentage was calculated.

Calculation of Efficacy

For each colony, diameter was measured twice by crossing method and the average was practicable. The colony growth inhibition rate was calculated as follows:

Colony growth inhibition rate %=(diameter of CK colony−diameter of target colony)×100/diameter of CK colony

The results are listed in Table 3.

Following the above method, as targets 17 pathogens originated from tropical plants were tested. The pathogens were Rhizopus sp., Aspergillus, Colletotrichum gloeosporioides Penz, Banana Fusarium wilt, Colletouichum coccodes , Hughas, B. heveas , Cucumber mosaic virusstrain banana, Banana Sigatoka Leaf Spot Disease, Colletotrichum gloeosporioides, Colletotrichum musae (Berk & Curt) Arx, Botryodiplodia theobromae, Cladosporium cucumerinum Ellis et Arthur, Periconia hevenae, Colletotrichum gloeosporioides, B. cinerea, Diplodia natalensis , and Curvularia.

The results are listed in Table 4.

3. In Vivo Micro-Screening Assay

To further study the fungicidal spectrum of the compound of Formula (I), indoor in vivo micro-screening assay were carried out on cucumber gray mold, cucumber scab, cucumber sclerotinia rot, cucumber brown spot, pepper phytophthora blight, and rice sheath blight. The results showed that the compound of Formula (I) had good prevention and treatment effect on cucumber gray mold, cucumber scab, cucumber brown spot, and rice sheath blight.

1) Target Strains

Cucumber scab strain: HX; cucumber brown spot strain: SDHGHB060509; cucumber sclerotinia rot strain: HGJH; pepper phytophthora blight strain: NSGP; and rice sheath blight strain: WK-1.

Cucumber gray mold strain had three different resistant strains: F109 (anti-benzimidazole and its derivatives, sensitive to N-phenylcarbamate, dicarboximide, and pyrimidine amine); Q503001 (sensitive to benzimidazole and its derivatives, N-phenylcarbamate, dicarboximide, and pyrimidine amine); and SC504002 (sensitive to benzimidazole and its derivatives as well as dicarboximide, anti-N-phenylcarbamate and anti-pyrimidine amine).

2) Medication and Inoculation

Medicament to be tested and medicament of control groups were prepared as needed in a sunny morning. Cucumber gray mold, cucumber scab, cucumber brown spot, cucumber sclerotinia rot, and rice sheath blight were medicated by spray, and after air dry, pathogens of cucumber scab and of cucumber brown spot were inoculated through spraying spore suspension respectively, pathogens of cucumber gray mold, of cucumber sclerotinia rot, and of rice sheath blight were inoculated through lawn foliage. Pepper phytophthora blight was medicated and inoculated by root-irrigation. Subsequently, all seedlings were cultured in wet environment.

3) Investigation

After the disease of control groups were serious, spot area, disease index, and the diseased seedling rate were investigated to calculate the efficacy. Cucumber scab, cucumber brown spot, and pepper phytophthora blight were investigated by grading method. The spot extension area of cucumber gray mold and of rice sheath blight was investigated. The diseased seedling rate of cucumber sclerotinia rot was investigated.

Disease index=Σ(amount of infected diseased leaves in a grade×relative value of the grade)×100/(total amount of leaves×9);

Diseased seedling rate (%)=(total amount of seedlings−amount of diseased seedlings)/total amount of seedlings×100%

Prevention and treatment effect (%)=(disease of control group−disease of experiment group)/disease of control group×100%

The results are listed in Table 5.

Acute Toxicity and Ames Test

Bioassay results showed that the compound of Formula (I) had good preventive effect on downy mildew and powdery mildew. The toxicological indexes of the compound were tested following the standard GB15670-1995 “Toxicological test methods of pesticides for registration” by Tongji Medical College, Huazhong University of Science and Technology. The results showed that the compound had low toxicity.

The results are listed in Table 6.

Environmental Assessment Test

After acute toxicity and Ames test had been finished, environmental toxicity test was conducted following “Guideline for Environmental Safety Evaluation of Chemical Pesticides” of Agriculture Ministry of China by Shenyang Chemical Research Institute/Supervision and Test Center for Pesticide Safety Evaluation and Quality Control. The results showed that the compound was environmentally friendly.

The results are listed in Table 7.

Enzyme Activity Test

Take NADH oxidase (NOX), succinate-cytochrome C oxidase (SCO), and Complex III as target enzymes, the inhibitory activity of the compound of Formula (I) was tested. The results showed that, the compound had much higher inhibitory activity on NOX, SCO, and Complex III than azoxystrobin which functioned as a control agent, particularly on Complex III.

a NOX is generally used to detect an inhibitor of Complex I and Complex III; SCO is generally used to detect an inhibitor of Complex II and Complex III. Thus, an inhibitor of Complex III should have inhibitory activity on the two enzymes.

b The test target is cytochrome bc1 complex of pig heart.

Preparation of Emulsifier Concentrate

Materials:

1) (E)-methyl 3-methoxy-2-(2-((5-methoxy-benzothiazol-2-ylthio)methyl)phenyl)acrylate. (prepared following Example 1) having 98% active ingredient. 2) Emulsifier: pesticide emulsifier No. 500 and Tween 80 (both from market) 3) Cosolvent: DMF, DMSO, and acetone (all from market) 4) Stabilizer: epoxidized soybean oil (from market) 5) Solvent: toluene and xylene (both from market)

Emulsifier Concentrate 1

To a beaker (1000 mL), 50 g (100% basis) of the compound of Formula (I), 150 g of DMF, and 50 g of acetone were added and stirred for dissolution. To the solution, 25 g of pesticide emulsifier No. 500 and 50 g of Tween 80, 20 g of epoxidized soybean oil, and 655 g of toluene were added. Thus, the emulsifier concentrate 1 having 5 wt. % of the compound of Formula (I) was obtained and further packed.

Emulsifier Concentrate 2

To a beaker (1000 mL), 75 g (100% basis) of the compound of Formula (I), 150 g of DMF, and 50 g of acetone were added and stirred for dissolution. To the solution, 50 g of pesticide emulsifier No. 500 and 50 g of Tween 80, 20 g of epoxidized soybean oil, and 605 g of toluene were added. Thus, the emulsifier concentrate 2 having 7.5 wt. % of the compound of Formula (I) was obtained and further packed.

Emulsifier Concentrate 3

To a beaker (1000 mL), 100 g (100% basis) of the compound of Formula (I), 150 g of DMF, and 150 g of acetone were added and stirred for dissolution. To the solution, 50 g of pesticide emulsifier No. 500 and 50 g of Tween 80, 20 g of epoxidized soybean oil, and 480 g of toluene were added. Thus, the emulsifier concentrate 3 having 10 wt. % of the compound of Formula (I) was obtained and further packed.

Field Efficacy Trial

On the basis of indoor activity evaluation, field efficacy trials of the compound of Formula (I) on cucumber downy mildew, cucumber powdery mildew, and melon powdery mildew were conducted. The results are listed in Tables 9-11.

While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.