Alkaloid Waltherione S analogue as well as synthesis method and application thereof
By simplifying the structural design of Waltherione A, pyridone and pyridine alkaloid Waltherione S analogs were synthesized, solving the problems of long synthetic routes and low yields in existing technologies. This provides safe and low-toxicity pesticides that are active against plant diseases and nematodes, and are suitable for agricultural production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-27
AI Technical Summary
The existing synthetic routes for the alkaloid Waltherione A are too long, have low yields, and are costly, making them difficult to apply to agricultural production. Furthermore, existing pesticides pose significant environmental hazards, necessitating the development of safer and less toxic pesticides for the control of plant diseases and pests.
By designing and synthesizing pyridone and pyridine alkaloid Waltherione S analogs, and by simplifying the structure of Waltherione A and using specific solvents and catalysts, compounds with broad-spectrum bactericidal activity against plant diseases were synthesized.
The synthesized Waltherione S analogues exhibit good activity against a variety of plant diseases and nematodes, providing a safe, low-toxicity, and innovative pesticide candidate suitable for agricultural production.
Smart Images
Figure CN121735835A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of Waltherione alkaloid compound technology, and particularly to a Waltherione S alkaloid analogue, its synthesis method, and its application. Background Technology
[0002] With the rapid expansion of the world's population, arable land resources are shrinking continuously. It is estimated that current crop yields need to be tripled to meet food demand. Against this backdrop, plant protection is crucial for increasing crop yields. Crops are frequently affected by pests, diseases, and weeds during their growth. The Food and Agriculture Organization of the United Nations (FAO) predicts that up to 40% of food crops are destroyed by plant diseases and pests each year, severely restricting the improvement of agricultural product yield and quality. As is well known, pesticides are one of the important measures for controlling pests, diseases, and weeds and protecting crops. With the rational use of pesticides to control harmful organisms, more than 65 million tons of food losses can be reduced annually.
[0003] Pesticides have a long history, with organic pesticides emerging and being widely used for the control of plant diseases and pests. The earliest organic pesticides were organochlorine insecticides, such as DDT and hexachlorocyclohexane (HCH). Subsequently, organophosphate insecticides appeared, such as methamidophos and parathion. Compared to organochlorine insecticides, organophosphate insecticides are cheaper and can control a wider range of pests. In addition, carbamate insecticides, such as carbaryl, have also been extensively developed. Although organically synthesized pesticides are highly effective and efficient, most are highly toxic and leave high residues, posing significant environmental hazards and even threatening ecological security. At the same time, the creation of new pesticides using natural products has gradually begun.
[0004] While some natural products possess novel structures and excellent biological activity, their structural complexity makes them difficult to synthesize and commercialize. This necessitates research into structural simplification and the exploration of active groups to obtain compounds that are easier to produce and exhibit better biological activity. Currently, over 60 alkaloids have been isolated from *Orychophragmus violaceus*, many of which possess novel structures and demonstrate good biological activity. For example, the alkaloid Waltherione A exhibits excellent nematicidal activity, with an EC50 of 0.27 μg / mL against the southern root-knot nematode. Antismone showed superior antifungal effects against eight plant pathogenic fungi compared to commercially available plant-derived fungicides (osthol, carvacrol, and eugenol). Both demonstrate good biological activity and are lead compounds for the development of new pesticides. However, both have relatively complex structures. The total synthetic route for Antismone has not yet been discovered, and while a synthetic route for Waltherione A has been reported, it is too long, with a very low yield of only 1.31%, and is time-consuming, labor-intensive, and costly, making it difficult to apply in agricultural production. Summary of the Invention
[0005] In view of this, this invention proposes an alkaloid Waltherione S analogue, its synthesis method, and its application. The aim is to seek more innovative pesticides with good efficacy, safety, and low toxicity for application in the control of plant diseases and pests.
[0006] The technical solution of this invention is implemented as follows: An alkaloid Waltherione S analogue, wherein the alkaloid Waltherione S analogue is a pyridone analogue and / or a pyridine analogue; the structural formula of the pyridone analogue is shown in Formula I, and the structural formula of the pyridine analogue is shown in Formula II: , In the formula, R is selected from one of phenyl, 2-chloro-4-methoxyphenyl, 4-chlorophenyl, 3,4,5-trifluorophenyl, 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 4-methoxyphenyl, thiophene, furanyl, ethylphenyl, and butyrylphenyl.
[0007] Furthermore, the structural formula for R is shown below: .
[0008] Furthermore, the pyridone analog (compound 4) is prepared by further reaction of compound 3; the pyridine analog (compound 5) is prepared by hydrolysis of the pyridone analog. The preparation method of compound 3 includes the following steps: S1. Using 4,6-dibromo-2-methyl-3-pyridinol as compound 1, an organic solvent was added, followed by the addition of anhydrous sodium carbonate and iodomethane. The mixture was stirred and reacted. After the reaction was completed, the reaction was quenched, extracted, the organic phase was dried, filtered, and the solvent was evaporated. The mixture was then passed through a silica gel column and eluted to obtain compound 2. S2 and compound 2 were dissolved in ultra-dry dimethyl sulfoxide, then NaH was added and stirred. Finally, benzyl alcohol was added dropwise and stirred to react. After the reaction was completed, the reaction was quenched, extracted, the organic phase was dried, filtered, and the solvent was evaporated. Then, the solution was separated by silica gel column chromatography to obtain compound 3. The structural formulas of compounds 1, 2, and 3 are shown below: .
[0009] Further, in step S1, the molar ratio of compound 1, anhydrous sodium carbonate, and iodomethane is 160-166:300-350:800-850; the organic solvent is N,N-dimethylformamide, and the molar ratio of compound 1 to N,N-dimethylformamide is 40-44g:150-180mL; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the reaction is quenched with water, and the molar ratio of compound 1 to water is 40-44g:100-120mL; the eluent for elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 8-12:1; the stirring speed is 600-800 r / min, and the time is 4-4.4 h. Further, in step S2, the ratio of compound 2 to ultra-dry dimethyl sulfoxide is 5-5.4 g: 30-34 mL; the molar ratio of compound 2, NaH, and benzyl alcohol is 16-20: 32-40: 16-21; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the reaction is quenched with water, and the ratio of compound 2 to water is 5-5.4 g: 30-34 mL; the eluent for elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 19-23:1; the stirring speed is 650-750 r / min, and the time is 30-34 min.
[0010] Furthermore, when R is selected from one of 4-chlorophenyl, 2-chloro-4-methoxyphenyl, 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 4-methoxyphenyl, furanyl, and cyclopropyl, the method for synthesizing the pyridone analogue includes the following steps: Compound 3 was dissolved in n-butanol, and then phenylboronic acid compound, palladium acetate, and tri-tert-butylphosphine tetrafluoroborate were added and stirred to react. NaOH aqueous solution was then added to react. After the reaction was completed, the reaction was quenched, extracted, the organic phase was dried, filtered, and the solvent was evaporated. Then, the mixture was passed through a silica gel column and eluted to obtain the target analog. The phenylboronic acid compound is selected from one of 4-chlorophenylboronic acid, 2-chloro-4-methoxy-phenylboronic acid, 3,4-(methylenedioxy)phenylboronic acid, 3,4-dimethoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 4-methoxyphenylboronic acid, and cyclopropylboronic acid. The molar ratio of compound 3, phenylboronic acid compound, palladium acetate, and tri-tert-butylphosphine tetrafluoroborate is 3~4:4.5~5.0:06~0.08:0.07~0.09; the volume ratio of compound 3 to n-butanol and NaOH aqueous solution is 1~2g:30~60mL:2.5~5.0mL; the NaOH aqueous solution has a mass-volume percentage of 5~6% w / v; the reaction is quenched with water, and the volume ratio of compound 3 to water is 1~2g:30~60mL; the eluent for elution is petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 15~20:1; the stirring speed is 650~750 r / min and the stirring time is 12~18 min.
[0011] Furthermore, when R is selected from phenyl, 3,4,5-trifluorophenyl, or thiophene, the method for synthesizing the pyridone analogue includes the following steps: Compound 3 was dissolved in 1,4-dioxane, and then phenylboronic acid compound, tetra-triphenylphosphine palladium and anhydrous potassium carbonate were added and stirred to react. After the reaction was completed, the reaction was quenched, extracted, the organic phase was dried, filtered and the solvent was evaporated. Then the solution was passed through a silica gel column and eluted to obtain the target analog. The phenylboronic acid compound is selected from one of phenylboronic acid, 3,4,5-trifluorophenylboronic acid, and 3-thiopheneboronic acid; The molar ratio of compound 3, phenylboronic acid compound, tetraphenylphosphine palladium, and anhydrous potassium carbonate is 3~4:4.5~5.0:0.15~0.18:3.0~3.5; the molar ratio of compound 3 and 1,4-dioxane is 1~2g:30~60mL; the reaction is quenched with water, and the molar ratio of compound 3 to water is 40~44g:30~60mL; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 15~20:1; the stirring reaction temperature is 120~130℃, the stirring speed is 650~750r / min, and the time is 24~25h.
[0012] Furthermore, when R is selected from ethylphenyl or butyrylphenyl, the method for synthesizing the pyridone analogue includes the following steps: Compound 3 was dissolved in anhydrous ethanol, and then an aromatic compound, tetra-triphenylphosphine palladium, anhydrous potassium carbonate and formic acid were added and stirred to react. After the reaction was completed, the reaction was quenched, extracted, the organic phase was dried, filtered and the solvent was removed under reduced pressure. Then the mixture was passed through a silica gel column and eluted to obtain the target analog. The aromatic compound is selected from styrene and 4-phenyl-1-butene; The molar ratio of compound 3, benzene aromatic compound, tetraphenylphosphine palladium, anhydrous potassium carbonate, and formic acid is 3~4:6.0~7.0:0.15~0.18:3.0~3.6:3~3.5; the reaction is quenched with water, and the ratio of compound 3 to water is 1~2g:30~60mL; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 13~20:1; the stirring reaction is carried out at a temperature of 120~130℃, a rotation speed of 650~750r / min, and a time of 24~25h.
[0013] Furthermore, the synthesis process of the pyridine analog is as follows: the pyridone analog is dissolved in a mixed acid, then the reaction is stirred, after the reaction is completed, the reaction is quenched, extracted, the organic phase is dried, filtered and the solvent is removed under reduced pressure, and then the target pyridine analog is obtained by silica gel column chromatography and elution separation.
[0014] Furthermore, the ratio of the pyridone analogue to the mixed acid is 0.4~0.5g:10~14mL; the mixed acid includes hydrochloric acid, trifluoroacetic acid, and acetic acid in a volume ratio of 5~10:1~2:5~10; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for elution is petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1~2:20~40; the stirring reaction is carried out at a speed of 460~550r / min, a temperature of 90~95℃, and a time of 12~14h.
[0015] Furthermore, compound 1 can be prepared by the following method: 2-Methyl-3-hydroxypyridine was added to the solvent to dissolve compound 1, and then N-bromosuccinimide was added and stirred to react. After the reaction was completed, the solid impurities were obtained by vacuum filtration, while the liquid system was retained. The solid impurities were washed, and the solvent was removed from the resulting solution under reduced pressure. Then, the solution was passed through a silica gel column and eluted to obtain compound 1.
[0016] Furthermore, the mass ratio of 2-methyl-3-hydroxypyridine to N-bromosuccinimide is 18~25:62~75; the solvent is acetonitrile, and the volume ratio of 2-methyl-3-hydroxypyridine to acetonitrile is 18~24g:150~180mL; the rinsing reagent is dichloromethane; the eluent for elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 4~6:1; the stirring reaction temperature is 90~95℃, the stirring speed is 480~520r / min, and the time is 1~1.4h.
[0017] Application of Waltherione S alkaloid analogues in the prevention and control of plant diseases.
[0018] Furthermore, the plant diseases mentioned include *Colletotrichum musae* (bacterial wilt of banana), *Fusarium graminearum* (bacterial scab of wheat), *Colletotrichum musae* (bacterial anthracnose of banana), *Phytophthora capsici* (bacterial phytophthora of pepper), *Botrytis cinerea* (bacterial gray mold of tomato), *Rhizoctonia solani* (bacterial sheath blight of rice), *Pyricularia oryzae Cav* (bacterial blast of rice), *Botryodiplodia theobromae* (bacterial stem rot of mango), and southern root-knot nematode.
[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention simplifies the structures of Waltherione A and Antidesmone to design and synthesize Waltherione S analogues, which are pyridine and pyridone alkaloids. The synthesized compounds exhibit broad-spectrum bactericidal activity against Fusarium wilt of banana, Fusarium head blight of wheat, Anthracnose of banana, Phytophthora capsulatum, Botrytis cinerea of tomato, Sheath blight of rice, Blast of rice, and stem rot of mango, and also show good nematicidal activity against root-knot nematodes. These compounds provide promising candidates for innovative, natural, safe, and low-toxicity pesticides, and are expected to be applied in agricultural production. Attached Figure Description
[0020] Figure 1 This is the 1H NMR spectrum of compound 2.
[0021] Figure 2 This is the carbon NMR spectrum of compound 2.
[0022] Figure 3 This is the 1H NMR spectrum of compound 3.
[0023] Figure 4 This is the carbon NMR spectrum of compound 3.
[0024] Figure 5 This is the 1H NMR spectrum of compound 4a.
[0025] Figure 6 This is the carbon NMR spectrum of compound 4a.
[0026] Figure 7 This is the 1H NMR spectrum of compound 4c.
[0027] Figure 8 This is the carbon NMR spectrum of compound 4c.
[0028] Figure 9This is the 1H NMR spectrum of compound 4d.
[0029] Figure 10 This is the carbon NMR spectrum of compound 4d.
[0030] Figure 11 The image shows the 1H NMR spectrum of compound 4i.
[0031] Figure 12 This is the carbon NMR spectrum of compound 4i.
[0032] Figure 13 The image shows the 1H NMR spectrum of compound 4j.
[0033] Figure 14 This is the carbon NMR spectrum of compound 4j.
[0034] Figure 15 This is the 1H NMR spectrum of compound 4l.
[0035] Figure 16 This is the carbon NMR spectrum of compound 4l.
[0036] Figure 17 This is the 1H NMR spectrum of compound 5f.
[0037] Figure 18 This is the carbon NMR spectrum of compound 5f.
[0038] Figure 19 The efficacy of compound 4j against live banana anthracnose fruit is shown in the diagram.
[0039] Figure 20 The diagram shows the in vivo control efficacy of compounds 4d, 4i, and 5f against southern root-knot nematodes; the arrows indicate obvious root knots. Detailed Implementation
[0040] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.
[0041] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.
[0042] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.
[0043] Example 1 - Synthesis of Compound 3 The synthesis of compound 3 includes the following steps: S1. Take a 500 mL three-necked flask, add compound 1 (4,6-dibromo-2-methyl-3-pyridinol) (43.49 g, 164.19 mmol), dissolve it with 150.00 mL DMF, then add anhydrous sodium carbonate (34.80 g, 328.38 mmol) and iodomethane (51.11 mL, 820.95 mmol), finally add a magnetic stir bar, place the apparatus on a magnetic stirrer, set the speed to 700 r / min, and react at room temperature for 4 h. After the reaction is completed by TLC monitoring, add 100.00 mL of water to quench the reaction, and perform three extractions with dichloromethane, each time using 50.00 mL. Dry the organic phase with anhydrous Na2SO4, filter, and evaporate the solvent by rotary evaporation. Then pass the solution through a silica gel column, elute with petroleum ether and ethyl acetate in a volume ratio of 10:1 to obtain target compound 2; Compound 2: pale yellow liquid, yield 92.31%, production 42.27 g. g.
[0044] S2. Take a 100 mL three-necked flask, add compound 2 (5.02 g, 18.00 mmol), dissolve it in 30.00 mL of ultradry DMSO, then add NaH (0.86 g, 35.83 mmol), add a magnetic stir bar, place the apparatus on a magnetic stirrer, set the speed to 700 r / min, and finally slowly add benzyl alcohol (1.98 mL, 19.04 mmol). Stir at room temperature for 30 min. After the reaction is completed by TLC monitoring, add 30.00 mL of water to quench the reaction, and extract three times with dichloromethane, each time using 10.00 mL. Dry the organic phase with anhydrous Na2SO4, filter, and evaporate the solvent. Then pass the solution through a silica gel column and elute with petroleum ether and ethyl acetate in a volume ratio of 20:1 to obtain target compound 3. Target compound 3: white solid, yield 93.55%, m.p 55.3℃, yield 5.17 g.
[0045] Example 2 - Preparation of Analogs The overall synthetic route for pyridone analogs and pyridine analogs is as follows: .
[0046] (a) Pyridone analogues The structural formulas of the synthesized pyridone analogues are shown in formulas 4a-4m below: .
[0047] (1) The synthesis method of compound 4c includes the following steps: Take a 100 mL three-necked flask, add compound 3 (1.00 g, 3.26 mmol), dissolve it in 30.00 mL of n-butanol, then add 4-chlorophenylboronic acid (0.76 g, 4.89 mmol), palladium acetate (15 mg, 2% mmol), and tri-tert-butylphosphine tetrafluoroborate (23 mg, 2.4% mmol). Finally, add a magnetic stir bar and place the apparatus on a magnetic stirrer at 700 r / min. Stir at room temperature for 15 min under nitrogen protection. Then, add 2.50 mL of 5% w / v NaOH aqueous solution (6.66 mmol) using a syringe. React at room temperature for 1 h. After the reaction is complete as monitored by TLC, quench the reaction with 30.00 mL of water and extract three times with 10.00 mL of dichloromethane each time. mL, the organic phase was dried with anhydrous Na2SO4, filtered and the solvent was evaporated, then passed through silica gel column and eluted with petroleum ether and ethyl acetate in a volume ratio of 15:1 to obtain the target compound 4c; compound 4c: white solid, yield 70.8%, melting point 83.7-84.8 ℃.
[0048] The synthesis methods of compounds 4b, 4e-4h, and 4j-4k are consistent with those of compound 4c; in the synthesis, 4-chlorophenylboronic acid is replaced with the same molar amounts of 2-chloro-4-methoxy-phenylboronic acid, 3,4-(methylenedioxy)phenylboronic acid, 3,4-dimethoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 4-methoxyphenylboronic acid, and cyclopropylboronic acid.
[0049] (2) The method for synthesizing compound 4a includes the following steps: Take a 100 mL three-necked flask, add compound 3 (1.00 g, 3.26 mmol), dissolve it in 30.00 mL of 1,4-dioxane, then add phenylboronic acid (0.60, 4.89 mmol), tetraphenylphosphine palladium (0.19 g, 0.16 mmol), and anhydrous potassium carbonate (0.45 g, 3.26 mmol). Finally, add a magnetic stir bar and place the apparatus on a magnetic stirrer at 700 r / min. React at 120 °C for 24 h under nitrogen protection. After the reaction is complete, add 30.00 mL of water and extract three times with dichloromethane, each time using 10.00 mL. mL, the organic phase was dried with anhydrous Na2SO4, filtered and the solvent was evaporated, then passed through silica gel column and eluted with petroleum ether and ethyl acetate in a volume ratio of 15:1 to obtain the target compound 4a; compound 4a: white solid, yield 68.3%, melting point 84.5-86.2 ℃.
[0050] The synthesis of compounds 4d and 4i is the same as that of 4a; during the synthesis, phenylboronic acid is replaced with the same molar amounts of 3,4,5-trifluorophenylboronic acid and 3-thiopheneboronic acid.
[0051] (3) The method for synthesizing compound 4l includes the following steps: Take a 100 mL three-necked flask, add compound 3 (1.00 g, 3.26 mmol), dissolve in 30.00 mL anhydrous ethanol, then add styrene (0.68, 6.52 mmol), tetrakis(triphenylphosphine)palladium (0.19 g, 0.16 mmol), anhydrous potassium carbonate (0.45 g, 3.26 mmol), and formic acid (0.12 mL, 3.18 mmol). Finally, add a magnetic stir bar and place the apparatus on a magnetic stirrer at 700 r / min. React at 80 °C for 24 h under nitrogen protection. After the reaction is complete, add 30.00 mL of water and extract three times with 10.00 mL of dichloromethane each time. Dry the resulting organic phase with anhydrous Na₂SO₄, filter, remove the solvent under reduced pressure, and then pass through a silica gel column. Elute with petroleum ether and ethyl acetate in a volume ratio of 15:1 to obtain the target compound 4l. Compound 4l: Yellow liquid, yield: 59.2%.
[0052] The synthesis of compound 4m is the same as that of 4l; in the synthesis, styrene is replaced with the same molar amount of 4-phenyl-1-butene.
[0053] (II) Synthesis of pyridine analogues (compound 5) The structural formulas of the synthesized pyridine analogues are shown in formulas 5a-5m below: .
[0054] The synthesis process of pyridine analogues is as follows: Take a 100 mL three-necked flask, add 0.50 g of the corresponding pyridinone analogue, and dissolve it in 11.00 mL of a mixed acid solution (HCl: TFA: HAC) with a volume ratio of 5:1:5. Add a magnetic stir bar and place the apparatus on a magnetic stirrer. Adjust the stirring speed to 500 r / min and react at 90 °C for 12 h. After the reaction is completed by TLC monitoring, add 10.00 mL of water and extract three times with dichloromethane, each time using 10.00 mL. Dry the organic phase with anhydrous Na2SO4, filter, remove the solvent under reduced pressure, and then pass the solution through a silica gel column. Elute with petroleum ether and ethyl acetate with a volume ratio of 1:20 to obtain the target pyridinone analogue. The synthetic route is as follows: .
[0055] Example 3 - Structural Characterization of Analogs 4,6-dibromo-3-methoxy-2-methylpyridine: 4,6-dibromo-3-methoxy-2-methylpyridine (compound 2): yellow liquid, yield 92.3%. 1 H NMR (400 MHz, Chloroform- d ) d 7.47 (s, 1H), 3.77 (s, 3H), 2.48 (s, 3H). 13 C NMR (100 MHz, Chloroform- d ) d 155.1, 151.8,134.7, 129.5, 128.8, 60.6, 19.6. HRESIMS m / z 281.8932 [M+H] + (calcd for C7H8Br2NO + , 281.8947). 4-(benzyloxy)-6-bromo-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-bromo-3-methoxy-2-methylpyridine (compound 3): white solid, yield 93.5%, melting point 55.3-56.8 °C. 1 H NMR (400 MHz, DMSO- d 6) d 7.52 – 7.41 (m, 4H), 7.40 – 7.34 (m, 1H), 7.29 (s, 1H), 5.25 (s,2H), 3.73 (s, 3H), 2.32 (s, 3H). 13 C NMR (100 MHz, DMSO) d 159.1, 152.9, 143.6,136.2, 134.9, 129.1, 128.8, 128.3, 111.8, 70.8, 60.4, 18.7. HRESIMS m / z 308.0280 [M+H] + (calcd for C 14 H 15 BrNO2 + , 308.0281).
[0056] 4-(benzyloxy)-3-methoxy-2-methyl-6-phenylpyridine: 4-(benzyloxy)-3-methoxy-2-methyl-6-phenylpyridine (4a) is a white solid with a yield of 68.3% and a melting point of 84.5-86.2 °C. 1 H NMR (400 MHz, DMSO- d 6) d 8.03 (d, J = 8.1 Hz, 2H), 7.62 – 7.46 (m, 4H), 7.46 – 7.32 (m, 5H), 5.36 (q, J = 6.3, 4.6 Hz, 2H), 3.78 (s, 3H), 2.44 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) d 157.89, 152.16, 151.86, 142.76, 139.25, 136.87, 129.08, 129.00,128.97, 128.59, 128.24, 126.96, 104.89, 70.21, 60.36, 40.24, 19.56. HRESIMS m / z 306.1486 [M+H] + (calcd for C 20 H 17 F3NO2 + , 306.1489).
[0057] 4-(benzyloxy)-6-(2-chloro-5-methoxyphenyl)-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-(2-chloro-5-methoxyphenyl)-3-methoxy-2-methylpyridine (4b): yellow liquid, yield 68.4%. 1 H NMR (400 MHz, DMSO- d 6) d 7.52 – 7.45 (m, 2H), 7.42 (ddd, J =7.6, 5.1, 2.0 Hz, 3H), 7.39 – 7.31 (m, 1H), 7.25 (s, 1H), 7.04 (d, J = 3.1 Hz, 1H), 6.99 (dd,J = 8.8, 3.1 Hz, 1H), 5.27 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 2.41 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d 158.4, 156.7, 152.0, 151.9, 142.7,140.3, 136.7, 131.1, 129.0, 128.6, 128.1, 122.8, 116.9, 116.0, 109.3, 70.2,60.4, 56.0, 19.4. HRESIMS m / z 370.1202 [M+H] + (calcd for C 21 H 21 ClNO3 + , 370.1204).
[0058] 4-(benzyloxy)-6-(4-chlorophenyl)-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-(4-chlorophenyl)-3-methoxy-2-methylpyridine (4c): white solid, yield 70.8%, melting point 83.7-84.8℃. 1 H NMR (400 MHz, DMSO- d 6) d 8.11 – 8.04 (m, 2H), 7.62 (s, 1H), 7.56 – 7.49(m, 4H), 7.48 – 7.40 (m, 2H), 7.40 – 7.33 (m, 1H), 5.36 (s, 2H), 3.78 (s,3H), 2.43 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d HRESIMS m / z 340.1096 [M+H] + (calcd for C20 H 19 ClNO2 + , 340.1099).
[0059] 4-(benzyloxy)-3-methoxy-2-methyl-6-(3,4,5-trifluorophenyl)pyridine: 4-(benzyloxy)-3-methoxy-2-methyl-6-(3,4,5-trifluorophenyl)pyridine (4d): white solid, yield 66.1%, melting point 87.1-88.3℃. 1 H NMR (400 MHz, DMSO- d 6) d 8.07 – 7.97 (m, 2H), 7.73 (s, 1H), 7.55 – 7.50 (m, 2H), 7.49 – 7.41 (m, 2H), 7.42 – 7.33 (m, 1H), 5.35 (s, 2H), 3.78(s, 3H), 2.43 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d 158.1, 152.2, 151.0 (ddd, J =224.2, 10.5, 5.0 Hz), 148.4, 143.4,139.4 (d, J = 228.0 Hz), 136.7, 135.9 (m),129.1, 128.7, 128.3, 111.2 (m), 105.3, 70.6, 60.4, 19.5. HRESIMS m / z 360.1207[M+H] + (calcd for C 20 H 17 F3NO2 + , 360.1206).
[0060] 6-(benzo[d][1,3]dioxol-5-yl)-4-(benzyloxy)-3-methoxy-2-methylpyridine: 6-(benzo[d][1,3]dioxol-5-yl)-4-(benzyloxy)-3-methoxy-2-methylpyridine (4e): white solid, yield 73.3%, melting point 65.2-66.1 °C. 1 H NMR (400 MHz, DMSO-d 6) d 7.62 – 7.57 (m, 2H),7.54 – 7.47 (m, 3H), 7.46 – 7.40 (m, 2H), 7.39 – 7.32 (m, 1H), 6.99 (d, J = 8.0Hz, 1H), 6.07 (s, 2H), 5.34 (s, 2H), 3.76 (s, 3H), 2.40 (s, 3H). 13 C NMR (100MHz, DMSO- d 6) d 157.7, 151.7, 151.6, 148.2, 148.2, 142.4, 136.9, 133.7, 129.1,128.6, 128.2, 121.0, 108.7, 107.2, 104.3, 101.7, 70.2, 60.3, 19.6. HRESIMS m / z 350.1385 [M+H] + (calcd forC 21 H 20 NO4 + , 350.1387).
[0061] 4-(benzyloxy)-6-(3,4-dimethoxyphenyl)-3-methoxy-2-methylpyridine: -(benzyloxy)-6-(3,4-dimethoxyphenyl)-3-methoxy-2-methylpyridine (4f): white solid ， Yield 81.9%, melting point 85.4-85.9 ℃. 1 H NMR (400 MHz, DMSO- d 6) d 7.60 – 7.51 (m, 4H), 7.48 (s, 1H), 7.44(dd, J = 8.3, 6.6 Hz, 2H), 7.40 – 7.33 (m, 1H), 7.02 (d, J = 8.1 Hz, 1H), 5.35(s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.76 (s, 3H), 2.42 (s, 3H). 13 C NMR (100MHz, DMSO- d 6) d 157.8, 152.1, 151.5, 149.9, 149.2, 142.3, 136.9, 132.1, 129.1,128.6, 128.3, 119.6, 112.0, 110.6, 104.3, 70.2, 60.4, 56.1, 56.0, 19.6.HRESIMS m / z 366.1703 [M+H] + (calcd for C 22 H 24 NO4 + , 366.1700).
[0062] 4-(benzyloxy)-6-(3,5-dimethoxyphenyl)-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-(3,5-dimethoxyphenyl)-3-methoxy-2-methylpyridine (4g): white solid, yield 78.5%, melting point 83.4-84.2 ℃. 1 H NMR (400 MHz, DMSO- d 6) d 7.56 – 7.49 (m, 3H), 7.47 – 7.40 (m,2H), 7.41 – 7.32 (m, 1H), 7.16 (d, J = 2.4 Hz, 2H), 6.54 (t, J = 2.3 Hz, 1H), 5.37 (s, 2H), 3.81 (s, 6H), 3.78 (s, 3H), 2.42 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d HRESIMS m / z 366.1701 [M+H] + (calcdfor C 22 H 24 NO4 + , 366.1700).
[0063] 4-(benzyloxy)-3-methoxy-6-(4-methoxyphenyl)-2-methylpyridine: 4-(benzyloxy)-3-methoxy-6-(4-methoxyphenyl)-2-methylpyridine (4h) white solid, yield 75.6%, melting point 67.3-69.1℃. 1 H NMR (400 MHz, DMSO- d 6) d 8.03 – 7.95 (m, 2H), 7.56 – 7.32 (m, 6H), 7.04 –6.97 (m, 2H), 5.34 (s, 2H), 3.78 (d, J = 16.6 Hz, 6H), 2.41 (s, 3H). 13 C NMR (101MHz, DMSO- d 6) d 160.20, 157.85, 152.02, 151.61, 142.24, 136.92, 131.84, 129.06, 128.56, 128.26, 128.22, 114.30, 103.95, 70.12, 60.34, 55.65, 19.54.
[0064] 4-(benzyloxy)-3-methoxy-2-methyl-6-(thiophen-2-yl)pyridine: 4-(benzyloxy)-3-methoxy-2-methyl-6-(thiophen-2-yl)pyridine (4i): yellow solid, yield 83.2%, melting point 85.5-86.4℃. 1 H NMR (400 MHz, DMSO- d 6) d 7.74 (dd, J = 3.6, 1.1 Hz, 1H), 7.61 – 7.50 (m,4H), 7.47 – 7.41 (m, 2H), 7.40 – 7.33 (m, 1H), 7.18 – 7.10 (m, 1H), 5.32 (s,2H), 3.75 (s, 3H), 2.37 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) dHRESIMS m / z 312.1052 [M+H] + (calcd for C 18 H 18 NO2S + , 312.1053).
[0065] 4-(benzyloxy)-6-(furan-2-yl)-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-(furan-2-yl)-3-methoxy-2-methylpyridine (4j): yellow solid, yield 83.6%, melting point 56.6-57.2 ℃. 1 HNMR (400 MHz, DMSO- d 6) d 8.23 (dd, J = 1.6, 0.8 Hz, 1H), 7.73 (t, J = 1.7 Hz, 1H),7.55 – 7.50 (m, 2H), 7.46 – 7.41 (m, 2H), 7.41 – 7.34 (m, 2H), 7.01 (dd, J =1.9, 0.8 Hz, 1H), 5.28 (s, 2H), 3.74 (s, 3H), 2.37 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d 157.7, 151.9, 147.2, 144.5, 142.2, 141.6, 136.8, 129.1, 128.6,128.4, 127.5, 109.4, 104.7, 70.2, 60.3, 19.4. HRESIMS m / z 296.1282 [M+H] + (calcdfor C 18 H 18 NO3 + , 296.1281).
[0066] 4-(benzyloxy)-6-cyclopropyl-3-methoxy-2-methylpyridine: 4-(benzyloxy)-6-cyclopropyl-3-methoxy-2-methylpyridine (4k): white solid, yield 65.3%, melting point 39.8-40.5 °C. 1 H NMR (400 MHz, DMSO-) d 6) d 7.51 – 7.45 (m, 2H), 7.45 – 7.39 (m, 2H), 7.39 – 7.31 (m,1H), 6.91 (s, 1H), 5.20 (s, 2H), 3.68 (s, 3H), 2.27 (s, 3H), 1.94 (tt, J = 7.5,5.3 Hz, 1H), 0.86 – 0.79 (m, 4H). 13 C NMR (100 MHz, DMSO- d 6) d 157.8, 157.2,151.2, 141.3, 136.9, 129.0, 128.5, 128.1, 105.3, 69.9, 60.2, 19.3, 17.1, 9.5.HRESIMS m / z 270.1488 [M+H] + (calcd for C 17 H 20 NO2 + , 270.1489).
[0067] 4-(benzyloxy)-3-methoxy-2-methyl-6-(1-phenylethyl)pyridine: 4-(benzyloxy)-3-methoxy-2-methyl-6-(1-phenylethyl)pyridine (4l): yellow liquid, yield: 59.2%. 1 H NMR (400MHz, DMSO- d 6) d 7.48 – 7.43 (m, 2H), 7.42 – 7.37 (m, 2H), 7.36 – 7.31 (m, 1H), 7.31 – 7.23 (m, 4H), 7.19 – 7.12 (m, 1H), 6.98 (s, 1H), 5.17 (s, 2H), 4.12(q, J= 7.2 Hz, 1H), 3.69 (s, 3H), 2.33 (s, 3H), 1.55 (d, J = 7.2 Hz, 3H). 13 C NMR (100 MHz, DMSO-) d 6) d 160.1, 157.4, 151.1, 145.8, 141.7, 136.7, 129.0, 128.7,128.6, 128.4, 127.8, 126.5, 106.2, 70.0, 60.2, 46.9, 21.3, 19.4. HRESIMS m / z 334.1800 [M+H] + (calcd for C 22 H 24 NO2 + , 334.1802).
[0068] 4-(benzyloxy)-3-methoxy-2-methyl-6-(3-phenylpropyl)pyridine: 4-(benzyloxy)-3-methoxy-2-methyl-6-(3-phenylpropyl)pyridine (4m): yellow liquid, yield 62.2%. 1 H NMR (400 MHz, DMSO- d 6) d 7.53 – 7.45 (m, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.38 – 7.32 (m, 1H), 7.32 – 7.24 (m, 2H), 7.23 – 7.14 (m, 3H), 6.91 (s, 1H), 5.19 (s, 2H), 3.71(d, J = 1.5 Hz, 3H), 2.60 (q, J = 7.1 Hz, 4H), 2.32 (t, J = 1.8 Hz, 3H), 2.01 –1.86 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d157.4, 157.3, 151.1, 142.5, 141.5,136.9, 129.0, 128.8, 128.7, 128.5, 128.2, 126.2, 106.7, 69.9, 60.2, 37.5,35.3, 31.5, 19.2. HRESIMS m / z 348.1957 [M+H] + (calcd for C 23 H 26 NO2 + , 348.1958).
[0069] 3-methoxy-2-methyl-6-phenylpyridin-4-ol: 3-methoxy-2-methyl-6-phenylpyridin-4-ol (5a) white solid, yield 67.4%, melting point 136.2-138.5℃. 1 H NMR (400 MHz, Methanol- d 4) d 7.82– 7.72 (m, 2H), 7.70 – 7.58 (m, 3H), 7.33 (s, 1H), 3.97 (s, 3H), 2.68 (s,3H). 13 C NMR (100 MHz, Methanol- d 4) d 165.0, 149.8, 148.3, 143.4, 131.45, 131.3,129.2, 127.7, 111.1, 60.3, 13.0. HRESIMS m / z 216.1024 [M+H] + (calcd forC 14 H 15 ClNO3 + , 216.1019).
[0070] 6-(2-chloro-5-methoxyphenyl)-3-methoxy-2-methylpyridin-4-ol: 6-(2-chloro-5-methoxyphenyl)-3-methoxy-2-methylpyridin-4-ol (5b): white solid, yield 76.7%, melting point 135.1-136.7 °C. 1 H NMR (400 MHz, Methanol- d 4) d 7.58 – 7.42 (m, 1H), 7.20 – 7.12 (m, 1H), 7.11 (d, J = 4.0 Hz, 1H), 6.82 (d, J = 4.7 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 2.51 (s, 3H). 13 C NMR (100 MHz, Methanol- d 4) d 168.1, 158.8, 146.2, 145.8,144.5, 132.3, 130.9, 123.3, 117.4, 116.3, 115.0, 59.7, 55.0, 12.7. HRESIMS m / z 280.0733 [M+H] + (calcd for C 14 H 15 ClNO3 + , 280.0735).
[0071] 6-(4-chlorophenyl)-3-methoxy-2-methylpyridin-4-ol: 6-(4-chlorophenyl)-3-methoxy-2-methylpyridin-4-ol (5c): white solid, yield 73.5%, melting point 176.2-177.5 °C. 1 H NMR (400MHz, Methanol- d 4) d 7.81 – 7.75 (m, 2H), 7.67 – 7.60 (m, 2H), 7.33 (s, 1H), 3.97 (s, 3H), 2.68 (s, 3H). 13 C NMR (100 MHz, Methanol- d 4) d 165.0, 148.5, 148.4,143.5, 137.6, 130.1, 129.4, 129.3, 111.3, 60.3, 13.0. HRESIMS m / z 250.0628 [M+H] + (calcd for C 13 H 13 ClNO2 +, 250.0629).
[0072] 3-methoxy-2-methyl-6-(3,4,5-trifluorophenyl)pyridin-4-ol: 3-methoxy-2-methyl-6-(3,4,5-trifluorophenyl)pyridin-4-ol (5d): white solid, yield 72.1%, melting point 180.2-181.3℃. 1 H NMR (400 MHz, Methanol- d 4) d 7.73 – 7.62 (m, 2H), 7.33 (s, 1H), 3.97 (s, 3H), 2.67 (s, 3H). 13 C NMR (100 MHz, Methanol- d 4) d 165.1, 153.0 – 145.0 (m), 148.7,146.3, 143.9, 128.4 – 127.2 (m), 113.7 – 112.6 (m), 112.0, 60.3, 13.1.HRESIMS m / z 270.0737 [M+H] + (calcd for C 13 H 11 F3NO2 + , 270.0736).
[0073] 6-(benzo[d][1,3]dioxol-5-yl)-3-methoxy-2-methylpyridin-4-ol: 6-(benzo[d][1,3]dioxol-5-yl)-3-methoxy-2-methylpyridin-4-ol (5e): white solid, yield 70.1%, melting point 54.8-55.7 °C. 1 H NMR (400 MHz, DMSO- d 6) d 7.37 (d, J = 1.8 Hz, 1H), 7.30 (dd, J =8.1, 1.9 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.95 (s, 1H), 6.12 (s, 2H), 3.78 (s, 3H), 2.43 (s, 3H). 13C NMR (100 MHz, DMSO- d 6) d 159.0, 158.7, 158.4, 158.1,148.8, 148.2, 121.5, 119.2, 116.2, 108.9, 107.6, 102.0, 59.7, 21.5. m / z 260.0918 [M+H] + (calcd for C 14 H 14 NO4 + , 260.0917).
[0074] 6-(3,4-dimethoxyphenyl)-3-methoxy-2-methylpyridin-4-ol: 6-(3,4-dimethoxyphenyl)-3-methoxy-2-methylpyridin-4-ol (5f): white solid, yield 74.3%, melting point 168.2-169.4 °C. 1 H NMR (400 MHz, DMSO- d 6) d 7.47 – 7.43 (m, 2H), 7.37 (dd, J = 8.4, 2.2 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 6H), 2.63 (s, 3H). 13 C NMR (100MHz, DMSO- d 6) d HRESIMS m / z 276.1229 [M+H] + (calcd forC 15 H 18 NO4 + , 276.1230).
[0075] 6-(3,5-dimethoxyphenyl)-3-methoxy-2-methylpyridin-4-ol: 6-(3,5-dimethoxyphenyl)-3-methoxy-2-methylpyridin-4-ol (5g): white solid, yield 69.0%, melting point 162.8-163.9 ℃. 1 H NMR (400 MHz, Methanol- d 4) d 7.25 (s, 1H), 6.89 (d, J = 2.2 Hz, 2H), 6.73 (d, J =2.3 Hz, 1H), 3.95 (s, 3H), 3.87 (s, 6H), 2.64 (d, J = 5.6 Hz, 3H). 13 C NMR (100MHz, Methanol- d 4) d 165.5, 161.7, 149.5, 147.7, 143.6, 133.3, 111.3, 105.7,102.5, 60.2 54.8, 12.9. HRESIMS m / z 276.1231 [M+H] + (calcd for C 15 H 18 NO4 + ,276.1230).
[0076] 3-methoxy-6-(4-methoxyphenyl)-2-methylpyridin-4-ol: 3-methoxy-6-(4-methoxyphenyl)-2-methylpyridin-4-ol (5h): white solid, yield 76.2%, melting point 165.2-166.3℃. 1 H NMR (400MHz, Methanol- d 4) d 7.59 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.57 (s,1H), 3.83 (d, J = 20.0 Hz, 6H), 2.42 (s, 3H). 13 C NMR (100 MHz, Methanol- d 4) d 161.53, 161.53, 147.80, 147.80, 145.53, 145.53, 128.27, 125.64, 114.13, 113.25, 58.78, 58.78, 54.54, 12.25.
[0077] 3-methoxy-2-methyl-6-(thiophen-2-yl)pyridin-4-ol: 3-methoxy-2-methyl-6-(thiophen-2-yl)pyridin-4-ol (5i): yellow solid, yield 73.2%, melting point 170.4-171.5 °C. 1 H NMR (400MHz, DMSO- d 6) d 7.52 (d, J = 14.9 Hz, 2H), 7.13 (d, J = 23.7 Hz, 2H), 3.73 (s, 3H), 2.34 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d 157.1, 152.2, 147.6, 145.2, 142.2,128.6, 127.8, 124.2, 106.0, 60.2, 19.3. HRESIMS m / z 222.0585 [M+H] + (calcd forC 11 H 12 NO2S + , 222.0583).
[0078] 6-(furan-2-yl)-3-methoxy-2-methylpyridin-4-ol: 6-(furan-2-yl)-3-methoxy-2-methylpyridin-4-ol (5j): yellow solid, yield 72.0%, melting point 88.4-89.1 ℃. 1 H NMR (400 MHz, DMSO- d 6) d 8.31 – 8.20 (m, 1H), 7.78 (t, J = 1.8 Hz, 1H), 6.95 (dd, J= 2.0, 0.9Hz, 1H), 6.70 (s, 1H), 3.72 (s, 3H), 2.31 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) d 158.7, 158.4, 144.9, 144.1, 141.9, 122.9, 116.1, 111.4, 109.2, 59.5, 15.7.HRESIMS m / z 206.0810 [M+H] + (calcd for C 11 H 12 NO3 + , 206.0812).
[0079] 6-cyclopropyl-3-methoxy-2-methylpyridin-4-ol: 6-cyclopropyl-3-methoxy-2-methylpyridin-4-ol (5k): white solid, yield 69.1%, melting point 157.5-158.9 °C. 1 H NMR (400 MHz, Methanol-) d 4) d 6.65 (s, 1H), 3.88 (s, 3H), 2.57 (s, 3H), 2.18 (tt, J = 8.4, 5.0Hz, 1H), 1.35 – 1.23 (m, 2H), 1.08 – 0.95 (m, 2H). 13 C NMR (100 MHz, Methanol- d 4) d 165.1, 155.5, 146.7, 142.3, 106.5, 60.2, 12.8, 12.6, 9.5. HRESIMS m / z 180.1018 [M+H] + (calcd for C 10 H 14 NO2 + , 180.1019).
[0080] 3-methoxy-2-methyl-6-(1-phenylethyl)pyridin-4-ol: 3-methoxy-2-methyl-6-(1-phenylethyl)pyridin-4-ol (5l): yellow liquid, yield 74.0%. 1 H NMR (400 MHz, Methanol- d 4) d 7.41– 7.33 (m, 2H), 7.33 – 7.25 (m, 3H), 6.93 (s, 1H), 4.36 (q, J = 7.2 Hz, 1H), 3.88 (s, 3H), 2.54 (s, 3H), 1.69 (s, 3H). 13 C NMR (100 MHz, Methanol- d 4) d 166.3,155.8, 146.9, 143.0, 141.1, 128.8, 127.3, 127.1, 110.8, 59.9, 42.3, 19.3,12.7. HRESIMS m / z 244.1329 [M+H] + (calcd for C 15 H 18 NO2 + , 244.1332).
[0081] 3-methoxy-2-methyl-6-(3-phenylpropyl)pyridin-4-ol: 3-methoxy-2-methyl-6-(3-phenylpropyl)pyridin-4-ol (5m): yellow liquid, yield 72.9%. 1 H NMR (400 MHz, Methanol- d 4) d 7.29– 7.23 (m, 2H), 7.21 – 7.13 (m, 3H), 6.91 (s, 1H), 3.87 (s, 3H), 2.84 – 2.78(m, 2H), 2.73 (t, J = 7.4 Hz, 2H), 2.51 (s, 3H), 2.10 – 2.00 (m, 2H). 13 C NMR (100 MHz, Methanol-) d 4) d165.8, 152.7, 146.5, 142.7, 140.9, 128.1, 125.6,111.8, 59.9, 34.7, 32.0, 30.3, 12.7. HRESIMS m / z 258.1486 [M+H] + (calcd forC 16 H 20 NO2 + , 258.1489).
[0082] Example 4 - Antibacterial Activity of Analogs The antibacterial activity of the analogue against eight plant pathogens was determined, including *Fusarium wiltii*, the causal agent of banana wilt. Colletotrichum musae ), wheat scab ( Fusarium graminearum Banana anthracnose bacteria ( Colletotrichum musae ), Phytophthora capsici ( Phytophthora capsici ), tomato gray mold ( Botrytis cinerea Rice sheath blight pathogen ( Rhizoctonia solani Rice blast fungus ( Pyricularia oryzae Cav Mango stem rot fungus ( Botryodiplodia theobromae ).
[0083] The in vitro antibacterial activity of the target compound was determined using the mycelial growth rate method. 15 mg of the target analog was weighed using a 0.01 g / mL balance and dissolved in 0.5 mL of DMF. After the solution became clear and transparent, the dissolved analog was added to 300 mL of PDA medium and shaken well to prepare a PDA medium containing 50 μg / mL of the analog. A PDA medium with 0.5 mL of distilled water was used as a blank control, DMF without 0.2 g / mL of the agent was used as a solvent control, and fluopyram at 50 μg / mL was used as a positive control. The media were poured into 9 cm petri dishes, with three replicates for each treatment. After the PDA solidified, the corresponding fungal mycelium was inoculated and the dishes were sealed with sealing film. The entire process was performed aseptically. The inoculated PDA medium was incubated at 25 °C for 2–5 days. When the colony diameter in the petri dishes reached 6 cm, it was measured using a ruler using the cross-hatching method. The average value was taken, and the inhibition rate was calculated. The results are shown in Table 1 below.
[0084] .
[0085] Note: The diameter of the mushroom cake is 5 mm.
[0086] Table 1 Antibacterial activity of analogues
[0087] Note: Po Rice blast fungus; Rs. Rice sheath blight pathogen; Bc Tomato gray mold pathogen; BT Mango stem rot fungus; Cm Banana anthracnose bacteria; Fg Fusarium graminearum, the pathogen that causes wheat blight; Fo Banana wilt pathogen; PC Phytophthora capsici, the pathogen of the pepper blight; Fluopyram: fluopyram.
[0088] As shown in Table 1, pyridone analogs and pyridine analogs exhibited varying degrees of antibacterial activity against eight plant pathogens.
[0089] Example 5 - Fruit control experiment of Waltherione S analogue 4j alkaloid Compound 4j, which showed good in vitro bactericidal activity against *Anthracnose* in bananas, was tested for its fruit-based efficacy. First, the banana surface was washed with water, then sterilized with 75% ethanol. Compound 4j was dissolved in DMF to prepare a stock solution. After the solution became clear, it was diluted with sterile water containing 0.5% Tween 80 to 100 µg / mL and 200 µg / mL, respectively. Sterile water containing 0.5% Tween 80 was used as a blank control (CK), Tween water containing an equal volume of solvent was used as a solvent control, and azoxystrobin (200 µg / mL) was used as a positive control. The bananas were then soaked in the prepared solution for 5 minutes and allowed to air dry. Finally, an anthracnose spore suspension (5-8 × 10⁻⁶) was added to the solution. 6 The treatment was evenly sprayed onto the surface of bananas (each banana per ml). The treated bananas were stored in an incubator at 28°C and 80% RH. Each treatment consisted of three replicates, with three bananas per replicate. Disease incidence was assessed after 5 days. Grading criteria and efficacy calculations are shown below, and the results are presented in Table 2. Figure 19 As shown.
[0090] The grading standards are as follows: Grade 0: No lesions; Grade 1: Lesions cover less than 5% of the fruit area; Grade 3: Lesions cover 6%-10% of the fruit area; Grade 5: Lesions cover 11%-25% of the fruit area; Grade 7: Lesions cover 26%-50% of the fruit area; Grade 9: Lesions cover more than 50% of the fruit area.
[0091] The efficacy calculation is as follows: .
[0092] Table 2. Protective efficacy of compound 4j on banana fruit.
[0093] Note: Lowercase letters represent the results of one-way ANOVA between different groups at the same time point (Duncan analysis P≤0.05). a, b, c, d indicate that there are significant differences between different groups, and a, b, c, d are labeled in descending order of inhibition rate.
[0094] From Table 2 and Figure 19 The results showed that the control efficacy of banana fruit treated with 200 μg / mL compound 4j was 39.6%, slightly lower than that of banana fruit treated with 200 μg / mL azoxystrobin (54.3%), while the control efficacy of banana fruit treated with 200 μg / mL compound 4j was 61.7%, slightly higher than that of banana fruit treated with 200 μg / mL azoxystrobin.
[0095] Example 6 - Nematicidal Activity of Alkaloid Waltherione S Analog The assay was performed using the immersion method. 2.5 mg of the test compound was weighed using a 0.01 g / mL balance and added to DMF (0.5 mL) to prepare a 5000 μg / mL stock solution. The concentration of the test compound was then diluted to 400 μg / mL with sterile water containing 0.5% Tween 80. Simultaneously, using *Sinomenium acutum* J2 as the test sample, nematodes were prepared to a concentration of 1000 nematodes / mL. A clean 24-well plate was prepared, with 100 µL of nematode solution and 100 µL of the test compound solution added to each well. The corrected mortality rate of the target analog at a concentration of 200 μg / mL was determined. DMF without 0.2 g / mL was used as a solvent control, and 50 μg / mL avermectin was used as a positive control. Each treatment was repeated in triplicate. The 24-well plates were incubated at 25 °C, and the number of dead and live nematodes was counted at 24 h, 48 h, and 72 h. The corrected mortality rates at 24 h, 48 h, and 72 h were calculated, and the results are shown in Table 3 below.
[0096] Mortality rate (%) = %; Corrected mortality rate (%) = % Table 3. In vitro nematicidal activity of analogs against southern root-knot nematodes.
[0097] As shown in Table 3, the prepared analogues exhibited varying degrees of insecticidal activity against nematodes.
[0098] Compounds 4d, 4i, and 5f, which exhibited good in vitro nematicidal activity, were selected for pot-based efficacy experiments. Pepper seedlings of uniform growth were transplanted into prepared 210 mm × 170 mm pots, one seedling per pot. Compounds 4d, 4i, and 5f were prepared at concentrations of 200 μg / mL and 500 μg / mL. Seven days after transplanting, 3-5 holes were drilled around the root zone of each plant, 5-7 cm deep, and the prepared solutions were poured in. Each plant received 10 mL of the test solution. A 10 μg / mL abamectin solution was used as a positive control, and water was used as a control solution. Each treatment was replicated five times. The day after root irrigation, 3-5 more holes were drilled around the root zone of each pepper plant, 6-8 cm deep, and approximately 800 second-instar nematodes were inoculated into each plant. Forty-five days later, the number of root knots produced on the pepper roots was counted, and the control effect was calculated. The results are shown in Table 4 below. Figure 20 As shown.
[0099] 00%.
[0100] Table 4. Results of pot experiment on the control efficacy of the target compound against southern root-knot nematode.
[0101] Note: Lowercase letters represent the results of one-way ANOVA between different groups at the same time point (Duncan analysis P≤0.05).
[0102] a, b, c, d, e, f, g indicate significant differences between different groups, and a, b, c, d, e, f, g are labeled in descending order of inhibition rate.
[0103] As shown in Table 4, Figure 20 As shown, the potted plant control efficacy of compounds 4d, 4i, and 5f against southern root-knot nematodes increased with increasing concentration. Among them, compound 4i showed potted plant control efficacy of 42.10% and 72.33% against southern root-knot nematodes at concentrations of 200 μg / mL and 500 μg / mL, respectively. The nematicidal activity of compound 4i at a concentration of 500 μg / mL was slightly better than that of the positive control avermectin at a concentration of 10 μg / mL. The control efficacies of compounds 4d and 5f at concentrations of 200 μg / mL and 500 μg / mL were 21.05%, 47.37%, 31.71%, and 55.69%, respectively.
[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. An alkaloid analogue of Waltherione S, characterized in that, The alkaloid Waltherione S analog is a pyridone analog and / or a pyridine analog; the structural formula of the pyridone analog is shown as formula I, and the structural formula of the pyridine analog is shown as formula II: ， In the formula, R is selected from phenyl, 2-chloro-4-methoxyphenyl, 4-chlorophenyl, 3,4,5-trifluorophenyl, 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 4-methoxyphenyl, thienyl, furanyl, ethylphenyl, butylphenyl.
2. The process for the synthesis of alkaloid analogues of the Waltherione S class as defined in claim 1, characterized by that, The pyridone analog is prepared by further reaction of compound 3; and the pyridine analog is prepared by hydrolysis reaction of the pyridone analog. The preparation method of the compound 3 comprises the following steps: S1, taking 4,6-dibromo-2-methyl-3-pyridinol as compound 1, adding an organic solvent, then adding anhydrous sodium carbonate and iodomethane for stirring reaction, after the reaction is completed, quenching the reaction, extraction, drying the organic phase, filtering and then rotary evaporation of the solvent, followed by silica gel column chromatography, elution separation to obtain compound 2; S2, dissolving compound 2 in super-dry dimethyl sulfoxide, then adding NaH for stirring, finally adding benzyl alcohol dropwise for stirring reaction, after the reaction is completed, quenching the reaction, extraction, drying the organic phase, filtering and then rotary evaporation of the solvent, followed by silica gel column chromatography, elution separation to obtain compound 3.
3. The synthesis method of the alkaloid Waltherione S analog according to claim 2, characterized in that, In step S1, the molar ratio of the compound 1, anhydrous sodium carbonate and iodomethane is 160-166:300-350:800-850; the organic solvent is N,N-dimethylformamide, and the usage ratio of the compound 1 and N,N-dimethylformamide is 40-44g:150-180mL; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the reaction is quenched with water, and the usage ratio of the compound 1 and water is 40-44g:100-120mL; the eluent for elution separation is petroleum ether and ethyl acetate, and the volume usage ratio of petroleum ether and ethyl acetate is 8-12:1; the stirring speed is 600-800r / min, and the stirring time is 4-4.4h; In step S2, the usage ratio of the compound 2 and super-dry dimethyl sulfoxide is 5-5.4g:30-34mL; the molar ratio of the compound 2, NaH and benzyl alcohol is 16-20:32-40:16-21; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the reaction is quenched with water, and the usage ratio of the compound 2 and water is 5-5.4g:30-34mL; the eluent for elution separation is petroleum ether and ethyl acetate, and the volume usage ratio of petroleum ether and ethyl acetate is 19-23:1; the stirring speed is 650-750r / min, and the stirring time is 30-34min.
4. The process for the synthesis of alkaloid Waltherione S analog according to claim 2, characterized by, When R is selected from one of 4-chlorophenyl, 2-chloro-4-methoxyphenyl, 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 4-methoxyphenyl, furanyl, cyclopropyl, the synthesis method of the pyridinone analogues comprises the following steps: Compound 3 is dissolved in n-butanol, and then a benzene boronic acid compound, palladium acetate and tri-tert-butylphosphonium tetrafluoroborate are added for stirring reaction, and then an aqueous NaOH solution is added for reaction; after the reaction is completed, the reaction is quenched, extracted, and the organic phase is dried, filtered and then the solvent is rotary dried, followed by silica gel column chromatography, and then the target analogues are separated by elution; The benzene boronic acid compound is selected from one of 4-chlorobenzenboronic acid, 2-chloro-4-methoxy-benzenboronic acid, 3,4-(methylenedioxy)benzenboronic acid, 3,4-dimethoxybenzenboronic acid, 3,5-dimethoxybenzenboronic acid, 4-methoxybenzenboronic acid, cyclopropylboronic acid; The molar ratio of the compound 3, the benzene boronic acid compound, palladium acetate, tri-tert-butylphosphonium tetrafluoroborate is 3-4: 4.5-5.0: 06-0.08: 0.07-009; the dosage ratio of the compound 3, n-butanol and the aqueous NaOH solution is 1-2g: 30-60mL: 2.5-5.0mL; the mass percentage of the aqueous NaOH solution is 5-6% w / v; the reaction is quenched with water, and the dosage ratio of the compound 3 and water is 1-2g: 30-60mL; the eluent for separation by elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 15-20: 1; the stirring speed is 650-750r / min, and the stirring time is 12-18min.
5. The process for the synthesis of alkaloid Waltherione S analog according to claim 2, characterized by, When R is selected from one of phenyl, 3,4,5-trifluorophenyl, thienyl, the synthesis method of the pyridinone analogues comprises the following steps: Compound 3 is dissolved in 1,4-dioxane, and then a benzene boronic acid compound, palladium tetraphenylphosphonium and anhydrous potassium carbonate are added for stirring reaction; after the reaction is completed, the reaction is quenched, extracted, and the organic phase is dried, filtered and then the solvent is rotary dried, followed by silica gel column chromatography, and then the target analogues are separated by elution; The benzene boronic acid compound is selected from one of benzene boronic acid, 3,4,5-trifluorobenzene boronic acid, 3-thiophene boronic acid; The molar ratio of the compound 3, the benzene boronic acid compound, palladium tetraphenylphosphonium and anhydrous potassium carbonate is 3-4: 4.5-5.0: 0.15-0.18: 3.0-3.5; the dosage ratio of the compound 3 and 1,4-dioxane is 1-2g: 30-60mL; the reaction is quenched with water, and the dosage ratio of the compound 3 and water is 40-44g: 30-60mL; the solvent for extraction is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for separation by elution is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 15-20: 1; the stirring temperature is 120-130℃, the stirring speed is 650-750r / min, and the stirring time is 24-25h.
6. The synthesis method of the alkaloid Waltherione S analogue according to claim 2, characterized in that, When R is selected from ethylphenyl, butylphenyl, one of the butylphenyl, the pyridone analog synthesis method comprises the following steps: Compound 3 is dissolved in anhydrous ethanol, and then aromatic compound, tetrakis triphenylphosphine palladium, anhydrous potassium carbonate and formic acid are added and stirred to react, after the reaction is completed, the reaction is quenched, extracted, the organic phase is dried, filtered, and then the solvent is removed under reduced pressure, followed by silica gel column, and elution separation to obtain the target analog; The aromatic compound is selected from one of styrene, 4-phenyl-1-butene; The molar ratio of compound 3, benzene aromatic compound, tetrakis triphenylphosphine palladium, anhydrous potassium carbonate, formic acid is 3-4:6.0-7.0:0.15-0.18:3.0-3.6:3-3.5; the reaction is quenched with water, and the amount ratio of compound 3 and water is 1-2g:30-60mL; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for elution separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 13-20:1; the stirring reaction temperature is 120-130℃, the rotation speed is 650-750r / min, and the time is 24-25h.
7. The process for the synthesis of alkaloid Waltherione S analog according to claim 2, characterized by, The synthesis process of the pyridine analog is as follows: dissolving the pyridone analog in mixed acid, then stirring to react, after the reaction is completed, the reaction is quenched, extracted, the organic phase is dried, filtered, and then the solvent is removed under reduced pressure, followed by silica gel column, and elution separation to obtain the target pyridine analog.
8. The process for the synthesis of alkaloid Waltherione S analog according to claim 7, characterized by, The amount ratio of the pyridone analog and the mixed acid is 0.4-0.5g:10-14mL; the mixed acid comprises hydrochloric acid, trifluoroacetic acid and acetic acid in a volume ratio of 5-10:1-2:5-10; the extraction solvent is dichloromethane; the drying reagent is anhydrous Na2SO4; the eluent for elution separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate is 1-2:20-40; the stirring reaction rotation speed is 460-550r / min, the temperature is 90-95℃, and the time is 12-14h.
9. The application of the alkaloid Waltherione S analog of claim 1 or the alkaloid Waltherione S analog synthesized by the synthesis method of claims 1-8 in preventing and treating plant diseases.
10. Use according to claim 9, characterized in that, The plant diseases include banana fusarium wilt, wheat scab, banana anthracnose, pepper pythium blight, tomato gray mold, rice sheath blight, rice blast, mango stem rot, southern root-knot nematode.