Melatonin derivative and application thereof in prevention and treatment of citrus penicillium rot

Abstract

The application discloses a melatonin derivative and application of the melatonin derivative in prevention and treatment of citrus penicillium disease. The compound has a structural formula shown in the following formula (I). Formula (I); the application has very strong inhibitory activity on pathogenicity of penicillium italicum and has good broad-spectrum properties. The application has inhibitory effects on fusarium graminearum, botrytis cinerea, sclerotinia sclerotiorum, cladosporium cladosporioides, pellicularia sasakii, lysobacter lactarius, fusarium oxysporum and fusarium oxysporum, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of novel pesticide compounds, specifically to a melatonin derivative and its application in controlling Penicillium citrus, or its application as a pesticide fungicide. Background Technology

[0002] With the continuous expansion of citrus planting scale and the constant increase in yield, the industry faces increasingly severe challenges, among which the impact of fungal diseases is particularly prominent. Fungal diseases such as Penicillium mold, canker, gray mold, and anthracnose not only seriously affect the appearance and internal quality of the fruit but also inhibit the normal growth of the fruit trees. Currently, chemical fungicides are the main means of controlling post-harvest diseases in citrus fruits, including imazalil, thiophanate-methyl, and others. These chemical pesticides are characterized by rapid effectiveness and significant control effects, thus effectively suppressing the spread of diseases in the short term. However, the pesticide residues, environmental pollution, and potential harm to human health caused by the long-term and large-scale application of chemical pesticides are increasingly attracting attention. At the same time, the increasing resistance of pathogens to pesticides further complicates disease control. Therefore, to ensure stable citrus yields and the safety of agricultural products, it is urgent to develop new, highly efficient, environmentally friendly, and low-toxicity pesticides.

[0003] Melatonin, an endogenous natural signaling molecule, is widely present in various organisms. In humans, it participates in regulating sleep cycles, slowing the aging process, and has physiological functions such as enhancing immunity, promoting digestion, and acting as a diuretic. In plants, melatonin also plays multiple regulatory roles, involving processes such as growth and development, improved tolerance to abiotic stresses such as drought and cadmium, enhanced disease resistance, improved fruit quality, and flowering induction. Currently, research on melatonin mainly focuses on its role in resisting biotic stress and its synergistic application with other agents, while reports on its direct use as an antibacterial substance for the control of plant fungal diseases are still relatively rare.

[0004] In recent years, researchers have used melatonin for the control of plant pathogenic fungi. However, there is limited research on the antibacterial effects of different derivatives obtained by structural modification using melatonin as a template. Studies have shown that the antibacterial activity of melatonin derivatives is closely related to the type and position of substituents on its indole and benzene rings, and reasonable modification can significantly enhance its antibacterial effect. This project, through systematic research, discovered melatonin derivatives with good antibacterial activity. These compounds are novel in structure, environmentally friendly, and exhibit good inhibitory activity against a variety of plant pathogenic fungi. Summary of the Invention

[0005] The purpose of this invention is to provide melatonin derivatives, preparation methods, and control of Penicillium italicum in citrus fruits. Penicillium italicum Application of ).

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a melatonin derivative having the structure shown in formula (I). Equation (Ⅰ), In equation (Ⅰ), R 1 Selected from H, methyl, phenyl; R 2 Selected from phenyl, 4-fluorophenyl, 3-fluorophenyl, 2-fluorophenyl, 3,5-difluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 4-trifluoromethylphenyl, 4-chlorophenyl, 3,5-dichlorophenyl, 3,4-dichlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-furanyl, 2-thienyl; R 3 Selected from H, halogen, methyl, methoxy, trifluoromethyl; R 3 It can be in any substitution position, either single or multiple substitution.

[0008] Preferably, the melatonin-containing derivative of the present invention is specifically: Compound M-1: R 1 For Ph, R 2 For Ph, R 3 For H; Compound M-2: R 1 CH3, R 2 For Ph, R 3 For H; Compound M-3: R 1 For H, R 2 For Ph, R 3 For H; Compound M-4: R 1 For H, R 2 It is 4-fluorophenyl, R 3 For H; Compound M-5: R 1 For H, R 2 It is 3-fluorophenyl, R 3 For H; Compound M-6: R 1 For H, R 2 It is 2-fluorophenyl, R 3 For H; Compound M-7: R 1 For H, R 2 It is 3,5-difluorophenyl, R 3 For H; Compound M-8: R 1 For H, R 2 It is 2,4-difluorophenyl, R 3 For H; Compound M-9: R1 For H, R 2 It is 3,4-difluorophenyl, R 3 For H; Compound M-10: R 1 For H, R 2 It is 4-trifluoromethylphenyl, R 3 For H; Compound M-11: R 1 For H, R 2 It is 4-chlorophenyl, R 3 For H; Compound M-12: R 1 For H, R 2 It is 3,5-dichlorophenyl, R 3 For H; Compound M-13: R 1 For H, R 2 It is 3,4-dichlorophenyl, R 3 For H; Compound M-14: R 1 For H, R 2 It is 4-methylphenyl, R 3 For H; Compound M-15: R 1 For H, R 2 It is 4-methoxyphenyl, R 3 For H; Compound M-16: R 1 For H, R 2 It is 2-furanyl, R 3 For H; Compound M-17: R 1 For H, R 2 It is 2-thienyl, R 3 For H; Compound M-18: R 1 For H, R 2 It is 3-fluorophenyl, R 3 5-F; Compound M-19: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 6-F; Compound M-20: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5.6-2F; Compound M-21: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-CF3; Compound M-22: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 7-CH3; Compound M-23: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-CH3; Compound M-24: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-MeO; Compound M-25: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-Br; Compound M-26: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-I; Compound M-27: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5.6-2Cl; The melatonin derivatives of this invention are synthesized using the following route:

[0009] In the above synthetic routes, the substituents of Formula I, Formula II, Formula III, Formula IV and Formula V are all defined in the same way as the corresponding groups in Formula (I) of the present invention.

[0010] In the above synthetic route, in step a, the synthesis method of formula II can be, for example, as follows: Place raw material I, ammonium acetate, and nitromethane in a 100 mL flask and react at 80-100 °C for 1-2 h until the reaction is complete. After the reaction is complete, remove unreacted nitromethane under reduced pressure, extract and separate the organic phases, combine them, dry and concentrate, and perform column chromatography to obtain the corresponding product II.

[0011] In the above synthetic route, in step b, the synthesis method of formula III can be, for example, as follows: intermediate II, zinc acetate, and cyclohexane are placed in a 100 mL flask, and indole or an indole derivative is added to the flask. The mixture is reacted at 50-75 °C for 10-12 h until the reaction is complete. After the reaction is complete, the cyclohexane solvent is removed under reduced pressure, the liquid and liquid phases are extracted and combined, dried and concentrated, and then subjected to column chromatography to obtain the corresponding product III.

[0012] In the above synthetic route, in step c, the synthesis method of formula IV can be, for example, as follows: under N2 atmosphere protection, lithium aluminum hydride is slowly added to the tetrahydrofuran solution of intermediate III at 0 °C, and then slowly raised to room temperature for 6 h; after the reaction is completed, water is added at 0 °C to quench the unreacted lithium aluminum hydride, and then diatomaceous earth is added for filtration, the organic phases are combined, dried and concentrated under reduced pressure to obtain an oily solid, and then purified by column chromatography to obtain product IV.

[0013] In the above synthetic route, in step d, the synthesis method of formula V can be, for example, as follows: Under a N2 atmosphere, a solution of triethylamine and intermediate IV in dichloromethane is added to a Shrek tube, and an anhydride is slowly added at low temperature. The mixture is then raised to room temperature and the reaction continues for 10-12 h until complete. A saturated aqueous solution of sodium bicarbonate is added to the system, the mixture is extracted, the organic phases are combined, dried, concentrated, and subjected to column chromatography to obtain product V.

[0014] This invention utilizes the melatonin derivatives described above at concentrations of 50-200 µM for the control of Penicillium italicum in citrus (…). Penicillium italicum Applications on ).

[0015] Selected important plant pathogenic fungi in agriculture: Fusarium graminearum ( Fusarium gramineae ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Corynebacterium tomatoense ( Corynespora cassicola ), Rhizoctonia solani ( Rhizoctonia solani Verticillium dahliae Verticillium wilt dahlias ), Watermelon Rhizoctonia solani ( Rhizoctonia solani (Cl) Fusarium oxysporum ( Fusarium oxysporum As a donor strain, the in vitro inhibitory activity of melatonin derivatives against this batch of fungi was tested using drug-containing culture medium at concentrations of 50-200 μM (preferably 50 μM).

[0016] The 50-200 µM concentration refers to any value or any interval within this concentration range.

[0017] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available.

[0018] Compared with the prior art, the present invention has the following advantages: 1. This type of melatonin derivative has environmentally friendly properties.

[0019] 2. Simple structure and easy preparation. The melatonin derivatives of the present invention have simple structural formulas and are easy to prepare.

[0020] 3. It has a strong broad spectrum of activity, with good inhibitory activity against all of them.

[0021] 4. Strong bactericidal activity; melatonin derivatives are effective against Penicillium italicum (…). Penicillium italicum It exhibits strong inhibitory activity. Compound M-20, in particular, has an EC50... 50 It can reach over 28 µM. Attached Figure Description

[0022] Figure 1 The results of treating *Penicillium italicum* with compounds M-1 to M-13 on day 7 in in vitro experiments are shown. A represents a schematic diagram of the plate experiment, and B represents the inhibition rate of *Penicillium italicum* after 7 days.

[0023] Figure 2 The results of in vitro experiments on Penicillium italicum treated with compounds M-14 to M-27 on day 7 are shown. A represents a schematic diagram of the plate experiment, and B represents the inhibition rate of Penicillium italicum after 7 days.

[0024] Figure 3 This diagram illustrates the effects of compound M-20 on various pathogenic fungi in an in vitro experiment. The different plant pathogenic fungi are: *Fusarium graminearum* (…). Fusarium gramineae ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Corynebacterium tomatoense ( Corynespora cassicola ), Rhizoctonia solani ( Rhizoctonia solani Verticillium dahliae Verticillium dahliae ), Watermelon Rhizoctonia solani ( Rhizoctonia nightshade (Cl) Fusarium oxysporum ( Fusarium oxysporum A is a schematic diagram of the plate experiment results, and B is the inhibition rate result. Detailed Implementation

[0025] Unless otherwise specified, all reagents used in the following experiments are commercially available. Unless otherwise specified, all experimental methods used in the following experiments are conventional methods in the field. The following are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the following embodiments and can have many variations. Therefore, any modifications or improvements made by those skilled in the art based on the disclosure of the present invention should fall within the scope of protection claimed by the present invention.

[0026] Example 1: Synthesis of compound M-3 N-(2-(1H-indol-3-yl)-2-phenylethyl)-1,1,1-trifluoromethanesulfonamide:

[0027] Step a, Synthesis of Intermediate II, trans-2-nitrostyrene: Commercially available benzaldehyde (1.5 g) and ammonium acetate (435 mg) were placed in a 100 mL flask, followed by the addition of nitromethane (15 mL) and reacted at 80 °C for 2 h. After the reaction was complete as monitored by TLC, unreacted nitromethane was removed under reduced pressure. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 25:1) to obtain a yellow solid, product II. Yield: 83%.

[0028] Step b, Synthesis of Intermediate III 3-(1-phenyl-2-nitroethyl)-1H-indole: Under a nitrogen atmosphere, intermediate II trans-2-nitrostyrene (1.8 g), zinc acetate (22 mg), and indole (1.2 g) were placed in a 100 mL flask, followed by the addition of 10 mL of cyclohexane. The reaction was carried out at 75 °C for 10 h. After the reaction was completed by TLC monitoring, the cyclohexane solvent was removed under reduced pressure, the mixture was extracted, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 10:1) to obtain a reddish-brown oily solid III, yield: 92%.

[0029] Step c, synthesis of intermediate IV 2-phenyl-2-(1H-indol-3-yl)ethylamine: Under N2 atmosphere, 5 mL of tetrahydrofuran solution of intermediate III (1.32 g) was placed in a three-necked flask, and then a tetrahydrofuran solution of lithium aluminum hydride (655 mg) was added dropwise at 0 °C. After the addition was complete, the mixture was allowed to rise to room temperature for 6 h. After intermediate IV was completely reacted by TCL monitoring, 5 mL of water was added to the system at 0 °C to quench excess lithium aluminum hydride. The mixture was then filtered with diatomaceous earth, and the combined organic phases were dried and concentrated under reduced pressure to obtain an oily solid. The product IV 2-phenyl-2-(1H-indol-3-yl)ethylamine was then purified by column chromatography (CH2Cl2:MeOH = 10:1), yielding 48%.

[0030] In step d, under a nitrogen atmosphere, 4 mL of dichloromethane solution of intermediate IV (500 mg) was added to a Shrek tube. Triethylamine (437 µL) was slowly added dropwise at room temperature, and the mixture was stirred for 10 min. Subsequently, 1 mL of DCM solution (425 µL) of trifluoromethanesulfonic anhydride was added dropwise under an ice bath. After the addition was complete, the mixture was brought to room temperature and reacted for 12 h. After the reaction of the starting material was monitored by TCL until it was complete, a saturated aqueous solution of sodium bicarbonate was added to the system. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:CH2Cl2=1:3) to obtain compound M-3, yield: 64%.

[0031] 1H NMR (600 MHz, CDCl3) δ 8.14 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.36 – 7.26 (m, 5H), 7.21 (t, J = 7.6 Hz, 1H), 7.11 –7.02 (m, 2H), 4.87 (t, J = 6.0 Hz, 1H), 4.49 (t, J = 7.8 Hz, 1H), 4.04 (m,1H), 3.85 (m, 1H). 13 C NMR (151 MHz, CDCl3) δ 140.08, 136.59, 129.11, 127.98, 127.54,126.30, 122.86, 122.80, 121.83, 120.74, 120.07, 119.12, 118.61, 115.04,111.45, 48.44, 43.80. 19 F NMR (565 MHz, CDCl3) δ -77.23. Example 2: Synthesis of compound M-5 1,1,1-trifluoro-N-(2-(3-fluorophenyl)-2-(1H-indol-3-yl)ethyl)methanesulfonamide:

[0032] Step a, Synthesis of Intermediate II, trans-3-(2-nitrovinyl)fluorobenzene: Commercially available 3-fluorobenzaldehyde (1.5 g) and ammonium acetate (372 mg) were placed in a 100 mL flask, followed by the addition of nitromethane (15 mL) and reaction at 80 °C for 2 h. After the reaction was complete as monitored by TLC, unreacted nitromethane was removed under reduced pressure. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 25:1) to obtain a yellow solid, product II. Yield: 79%.

[0033] Step b, Synthesis of Intermediate III 3-(1-(3-fluorophenyl)-2-nitroethyl)-1H-indole: Under a nitrogen atmosphere, intermediate II trans-3-(2-nitrovinyl)fluorobenzene (2 g), zinc acetate (18 mg), and indole (1.2 g) were placed in a 100 mL flask, followed by the addition of 10 mL of cyclohexane. The reaction was carried out at 75 °C for 10 h. After the reaction was completed by TLC monitoring, the cyclohexane solvent was removed under reduced pressure, the mixture was extracted, the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 10:1) to obtain a reddish-brown oily solid III, yield: 83%.

[0034] Step c, synthesis of intermediate IV 2-(1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine: Under N2 atmosphere, 5 mL of tetrahydrofuran solution of intermediate III (1.42 g) was placed in a three-necked flask, and then a tetrahydrofuran solution of lithium aluminum hydride (655 mg) was added dropwise at 0 °C. After the addition was complete, the mixture was allowed to rise to room temperature for 6 h. After intermediate IV was completely reacted by TCL monitoring, 5 mL of water was added to the system at 0 °C to quench excess lithium aluminum hydride. The mixture was then filtered with diatomaceous earth, and the combined organic phases were dried and concentrated under reduced pressure to obtain an oily solid. The product IV 2-(1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine was then purified by column chromatography (CH2Cl2:MeOH = 10:1), yielding 41%.

[0035] In step d, under a nitrogen atmosphere, 4 mL of dichloromethane solution of intermediate IV (500 mg) was added to a Shrek tube. Triethylamine (410 µL) was slowly added dropwise at room temperature, and the mixture was stirred for 10 min. Subsequently, 1 mL of DCM solution (422 µL) of trifluoromethanesulfonic anhydride was added dropwise under an ice bath. After the addition was complete, the mixture was brought to room temperature and reacted for 12 h. After the reaction of the starting material was monitored by TCL until it was complete, a saturated aqueous solution of sodium bicarbonate was added to the system. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:CH2Cl2=1:3) to give compound M-5, yield: 76%.

[0036] 1 H NMR (600 MHz, CDCl3) δ 8.11 (s, 1H), 7.34 (m, 2H), 7.24 (m, 1H), 7.15 (m, 1H), 7.07 – 6.98 (m, 3H), 6.94 – 6.86 (m, 2H), 4.83 (t, J = 6.0 Hz, 1H), 4.42 (t, J = 7.8 Hz, 1H), 3.92 (m, 1H), 3.77 (m, 1H). 13 C NMR (151 MHz, CDCl3) δ 162.94, 161.31, 141.77 (d, J = 6.7 Hz), 135.53, 129.52 (d, J = 8.3 Hz), 125.06, 122.59 (d, J = 2.8 Hz), 121.89,120.80, 119.14, 117.92, 113.85 (d, J = 21.7 Hz), 113.53, 113.22, 110.50,47.18, 42.52. 19 F NMR (565 MHz, CDCl3) δ -77.09 – -77.20 (m), -111.72 – -112.07 (m). Example 3: Synthesis of compound M-18 N-[2-(5-fluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethyl]-1,1,1-trifluoromethanesulfonamide

[0037] Step a, Synthesis of Intermediate II, trans-3-(2-nitrovinyl)fluorobenzene: Commercially available 3-fluorobenzaldehyde (1.5 g) and ammonium acetate (372 mg) were placed in a 100 mL flask, followed by the addition of nitromethane (15 mL) and reaction at 80 °C for 2 h. After the reaction was complete as monitored by TLC, unreacted nitromethane was removed under reduced pressure. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 25:1) to obtain a yellow solid, product II. Yield: 79%.

[0038] Step b, Synthesis of Intermediate III 5-fluoro-3-[1-(3-fluorophenyl)-2-nitroethyl]-1H-indole: Under a nitrogen atmosphere, intermediate II trans-3-(2-nitrovinyl)fluorobenzene (2 g), zinc acetate (18 mg), and 5-fluoroindole (1.35 g) were placed in a 100 mL flask, followed by the addition of 10 mL of cyclohexane. The reaction was carried out at 75 °C for 12 h. After the reaction was completed by TLC monitoring, the cyclohexane solvent was removed under reduced pressure, the liquid phases were extracted and combined, dried, concentrated, and subjected to column chromatography (PE:EA = 10:1) to obtain a reddish-brown oily solid III, yield: 89%.

[0039] Step c, synthesis of intermediate 2-(5-fluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine: Under N2 atmosphere, 5 mL of tetrahydrofuran solution of intermediate III (1.51 g) was placed in a three-necked flask, and then a tetrahydrofuran solution of lithium aluminum hydride (655 mg) was added dropwise at 0 °C. After the addition was complete, the mixture was allowed to rise to room temperature for 6 h. After intermediate IV was completely reacted by TCL monitoring, 5 mL of water was added to the system at 0 °C to quench excess lithium aluminum hydride. The mixture was then filtered with diatomaceous earth, and the combined organic phases were dried and concentrated under reduced pressure to obtain an oily solid. The product IV, 2-(5-fluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine, was purified by column chromatography (CH2Cl2:MeOH = 10:1) with a yield of 39%.

[0040] In step d, under a nitrogen atmosphere, 4 mL of dichloromethane solution of intermediate IV (500 mg) was added to a Shrek tube. Triethylamine (385 µL) was slowly added dropwise at room temperature, and the mixture was stirred for 10 min. Subsequently, 1 mL of DCM solution (371 µL) of trifluoromethanesulfonic anhydride was added dropwise under an ice bath. After the addition was complete, the mixture was brought to room temperature and reacted for 12 h. After the reaction of the starting material was monitored by TCL until it was complete, a saturated aqueous solution of sodium bicarbonate was added to the system. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:CH2Cl2=1:3) to give compound M-18, yield: 70%.

[0041] 1 H NMR (600 MHz, CDCl3) δ 8.20 (s, 1H), 7.37 – 7.29 (m, 2H), 7.16 (d,J = 2.6 Hz, 1H), 7.12 (m, 1H), 7.06 – 6.93 (m, 4H), 4.82 (t, J = 6.0 Hz, 1H), 4.43 (t, J = 7.8 Hz, 1H), 3.98 (m, 1H), 3.88 – 3.80 (m, 1H). 13C NMR (151 MHz, CDCl3) δ 164.03, 157.09, 142.41 (d, J = 6.7 Hz), 133.01, 130.67, 123.59 (d, J = 3.0 Hz), 123.35, 114.90, 114.77 (d, J = 5.0Hz), 114.65, 112.24 (d, J = 9.7 Hz), 111.60, 111.43, 104.06, 103.90, 48.09,43.47. 19 F NMR (565 MHz, CDCl3) δ -76.97 – -77.42 (m), -111.39 – -111.94 (m), -122.80 – -123.46 (m). Example 4: Synthesis of compound M-20 N-[2-(5,6-difluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethyl]-1,1,1-trifluoromethanesulfonamide:

[0042] Step a, Synthesis of Intermediate II, trans-3-(2-nitrovinyl)fluorobenzene: Commercially available 3-fluorobenzaldehyde (1.5 g) and ammonium acetate (372 mg) were placed in a 100 mL flask, followed by the addition of nitromethane (15 mL) and reaction at 80 °C for 2 h. After the reaction was complete as monitored by TLC, unreacted nitromethane was removed under reduced pressure. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 25:1) to obtain a yellow solid, product II. Yield: 79%.

[0043] Step b, Synthesis of Intermediate III 5,6-difluoro-3-[1-(3-fluorophenyl)-2-nitroethyl]-1H-indole: Under a nitrogen atmosphere, intermediate II trans-3-(2-nitrovinyl)fluorobenzene (2 g), zinc acetate (18 mg), and 5,6-difluoroindole (1.53 g) were placed in a 100 mL flask, followed by the addition of 10 mL of cyclohexane. The reaction was carried out at 75 °C for 10–12 h. After the reaction was completed by TLC monitoring, the cyclohexane solvent was removed under reduced pressure, the liquid phases were extracted and combined, dried, concentrated, and subjected to column chromatography (PE:EA = 10:1) to obtain a reddish-brown oily solid III, yield: 68%.

[0044] Step c, synthesis of intermediate 2-(5,6-difluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine: Under N2 atmosphere, 5 mL of tetrahydrofuran solution of intermediate III (1.60 g) was placed in a three-necked flask, and then a tetrahydrofuran solution of lithium aluminum hydride (655 mg) was added dropwise at 0 °C. After the addition was complete, the mixture was brought to room temperature for 6 h. After intermediate IV was completely reacted by TCL monitoring, 5 mL of water was added to the system under ice bath to quench excess lithium aluminum hydride. The mixture was then filtered with diatomaceous earth, and the combined organic phases were dried and concentrated under reduced pressure to obtain an oily solid. The product IV, 2-(5,6-difluoro-1H-indol-3-yl)-2-(3-fluorophenyl)ethylamine, was then purified by column chromatography (CH2Cl2:MeOH = 10:1) with a yield of 43%.

[0045] In step d, under a nitrogen atmosphere, 4 mL of dichloromethane solution of intermediate IV (500 mg) was added to a Shrek tube. Triethylamine (358 µL) was slowly added dropwise at room temperature, and the mixture was stirred for 10 min. Subsequently, 1 mL of DCM solution (345 µL) of trifluoromethanesulfonic anhydride was added dropwise under an ice bath. After the addition was complete, the mixture was brought to room temperature and reacted for 12 h. After the reaction was monitored by TCL until the starting material was completely reacted, a saturated aqueous solution of sodium bicarbonate was added to the system. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE: CH2Cl2 = 1:3) to give compound M-20, yield: 75%.

[0046] 1 H NMR (600 MHz, CDCl3) δ 8.19 (s, 1H), 7.35 (m, 1H), 7.18 (m, 1H), 7.13 – 7.04 (m, 2H), 7.04 – 6.97 (m, 2H), 6.94 (m, 1H), 4.83 (m, 1H), 4.40(t, J = 7.7 Hz, 1H), 3.96 (m, 1H), 3.82 (m, 1H). 13C NMR (151 MHz, CDCl3) δ 164.05, 162.41, 145.85, 142.19 (d, J = 6.6Hz), 131.50 (d, J = 10.3 Hz), 130.79 (d, J = 8.3 Hz), 123.59 (d, J = 3.0 Hz), 122.80 (d, J = 3.7 Hz), 121.46 (d, J = 7.1 Hz), 120.62, 114.91 (d, J = 8.9Hz), 114.73, 105.66 (d, J = 19.4 Hz), 99.54, 99.40, 48.13, 43.43. 19 F NMR (565 MHz, CDCl3) δ -76.57 – -77.81 (m), -111.46, -141.99, -145.70. Example 5: Synthesis of compound M-22 N-[2-(3-fluorophenyl)-2-(7-methyl-1H-indol-3-yl)ethyl]-1,1,1-trifluoromethanesulfonamide

[0047] Step a, Synthesis of Intermediate II, trans-3-(2-nitrovinyl)fluorobenzene: Commercially available 3-fluorobenzaldehyde (1.5 g) and ammonium acetate (372 mg) were placed in a 100 mL flask, followed by the addition of nitromethane (15 mL) and reaction at 80 °C for 2 h. After the reaction was complete as monitored by TLC, unreacted nitromethane was removed under reduced pressure. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 25:1) to obtain a yellow solid, product II. Yield: 79%.

[0048] Step b, synthesis of intermediate III 7-methyl-3-[1-(3-fluorophenyl)-2-nitroethyl]-1H-indole: Under a nitrogen atmosphere, intermediate II trans-3-(2-nitrovinyl)fluorobenzene (2 g), zinc acetate (18 mg), and 7-methylindole (1.31 g) were placed in a 100 mL flask, followed by the addition of 10 mL of cyclohexane. The reaction was carried out at 75 °C for 10–12 h. After the reaction was completed by TLC monitoring, the cyclohexane solvent was removed under reduced pressure, the mixture was extracted, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE:EA = 10:1) to obtain a reddish-brown oily solid III, yield: 74%.

[0049] Step c, synthesis of intermediate 2-(3-fluorophenyl)-2-(7-methyl-1H-indol-3-yl)ethylamine: Under N2 atmosphere, 5 mL of tetrahydrofuran solution of intermediate III (1.48 g) was placed in a three-necked flask, and then a tetrahydrofuran solution of lithium aluminum hydride (655 mg) was added dropwise at 0 °C. After the addition was complete, the mixture was brought to room temperature and reacted for 6 h. After intermediate IV was completely reacted by TCL monitoring, 5 mL of water was added to the system under ice bath to quench excess lithium aluminum hydride. The mixture was then filtered with diatomaceous earth, and the combined organic phases were dried and concentrated under reduced pressure to obtain an oily solid. The product IV, 2-(3-fluorophenyl)-2-(7-methyl-1H-indol-3-yl)ethylamine, was purified by column chromatography (CH2Cl2:MeOH = 10:1) with a yield of 48%.

[0050] In step d, under a nitrogen atmosphere, 4 mL of dichloromethane solution of intermediate IV (500 mg) was added to a Shrek tube. Triethylamine (364 µL) was slowly added dropwise at room temperature, and the mixture was stirred for 10 min. Subsequently, 1 mL of DCM solution (354 µL) of trifluoromethanesulfonic anhydride was added dropwise under ice bath conditions. After the addition was complete, the mixture was brought to room temperature and reacted for 12 h. After the reaction was monitored by TCL until the starting material was completely reacted, a saturated aqueous solution of sodium bicarbonate was added to the system. The mixture was extracted, separated, and the organic phases were combined, dried, concentrated, and subjected to column chromatography (PE: CH2Cl2 = 1:3) to give compound M-22, yield: 76%.

[0051] 1 H NMR (600 MHz, CDCl3) δ 8.10 (s, 1H), 7.33 – 7.24 (m, 2H), 7.11 (m,1H), 7.08 – 7.06 (m, 1H), 7.06 – 7.00 (m, 2H), 6.99 – 6.93 (m, 2H), 4.86 (t,J = 6.0 Hz, 1H), 4.47 (t, J = 7.8 Hz, 1H), 3.98 (m, 1H), 3.83 (m,1H), 2.48(s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.96, 162.32, 142.87 (d, J = 6.7 Hz), 136.20, 130.52 (d, J = 8.2 Hz), 125.64, 123.60 (d, J = 2.8 Hz), 123.41,121.58, 120.80, 120.40, 116.66, 114.94, 114.80, 114.67, 114.36, 48.19, 43.63. 19 F NMR (565 MHz, CDCl3) δ -76.99 – -77.46 (m), -111.77 – -112.10 (m). The synthesis principles of the other compounds in the examples are the same as those of the reactions described above.

[0052] The experimental methods and procedures for compounds M-1 and M-2 were the same as in Example 1, except that the indole in step b was replaced with 2-phenylindole and 2-methylindole, respectively, to obtain two products with the following structural formulas. The final yields of these two products were 63% and 57%, respectively.

[0053]

[0054] The experimental methods and procedures for compounds M-4, M-6, M-7, M-8, M-9, M-10, M-11, M-12, M-13, M-14, M-15, M-16, and M-17 were the same as in Example 2, except that 3-fluorobenzaldehyde in step a was replaced with p-fluorobenzaldehyde, 2-fluorobenzaldehyde, 3,5-difluorobenzaldehyde, 2,4-difluorobenzaldehyde, 3,4-difluorobenzaldehyde, p-trifluoromethylbenzaldehyde, p-chlorobenzaldehyde, 3,5-dichlorobenzaldehyde, 3,4-dichlorobenzaldehyde, p-methylbenzaldehyde, p-methoxybenzaldehyde, 2-thiophenealdehyde, and furfural, respectively, yielding 13 products with the following structural formulas. The final yields of these 13 products were 47%, 55%, 69%, 74%, 49%, 62%, 44%, 61%, 64%, 71%, 76%, 62%, and 81%, respectively.

[0055]

[0056] The experimental methods and procedures for compounds M-19, M-21, M-23, M-24, M-25, M-26, and M-27 were the same as in Example 3, except that the 5-fluoroindole in step b was replaced with p-6-fluoroindole, 5-(trifluoromethyl)indole, 5-methylindole, 5-methoxyindole, 5-bromoindole, 5-iodoindole, and 5,6-dichloroindole, respectively, yielding seven products with the following structural formulas. The final yields of these seven products were 58%, 64%, 69%, 77%, 53%, 75%, and 55%, respectively.

[0057] .

[0058] Comparative Example 1: The antibacterial activity of compound M-20 of this patent was compared with that of the prior art. Compounds disclosed in the invention patent of Teng Huailong, Cai Huanyu, et al. of Huazhong Agricultural University were selected as a comparison. Among them, compound N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-4-methylbenzenesulfonamide (structural formula below) showed an inhibition rate of only 5.99% against *Penicillium italicum* at a concentration of 100 µM. The activity was increased by more than 12 times. Therefore, the antibacterial activity of the prior art compound is significantly lower than that of the compound in this patent. Thus, the disclosed patent cannot destroy the novelty or inventiveness of this patent, and therefore cannot be used as a prior art document in this case.

[0059]

[0060] Comparative Example 2: To verify the technical effect of compound M-20 of this patent, a parallel comparative experiment was conducted using compounds from the invention patent of Teng Huailong, Cai Huanyu, et al. of Huazhong Agricultural University. At a test concentration of 100 µM, the prior art compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]-1,1,1-trifluoromethanesulfonamide (structural formula below) showed an inhibition rate of 17.08% against Penicillium italicum. The above results indicate that the antibacterial activity of the prior art compound is significantly lower than that of the compound of this patent.

[0061]

[0062] Comparative Example 3: The compound M-20 of this patent is compared with the compound disclosed in the invention patent of Teng Huailong, Cai Huanyu et al. of Huazhong Agricultural University. The compound N-[2-(6-trifluoromethyl-1H-indol-3-yl)ethyl]-1,1,1-trifluoromethanesulfonamide (structural formula below) showed an inhibition rate of only 20.32% against Penicillium italicum at a concentration of 100 µM. It can be seen that the antibacterial activity of the prior art compound is significantly lower than that of the compound of this patent. Therefore, the prior art patent cannot destroy the novelty or inventiveness of this patent, that is, it cannot be used as a prior art document in this case.

[0063]

[0064] The inhibitory activity of the prepared compounds against nine representative plant pathogenic fungi was determined.

[0065] The culture media that may be involved in this invention are as follows: Potato dextrose agar (PDA): Wash and peel the potatoes, then weigh 200 g of potatoes, cut them into small pieces, add water and boil until soft (boil for 20-30 minutes, until they can be pierced with a glass rod). Filter through four layers of gauze, add 20 g of D-glucose, stir well, cool slightly, then add water to make up to 1000 mL. Finally, add 15 g of agar and autoclave at 115 ℃ for 20 min.

[0066] The method for determining antibacterial activity is as follows: Culture of plant pathogenic fungi: *Penicillium* was inoculated onto PDA medium and cultured for 7 days in a constant temperature incubator at 27±0.1 ℃. Once the mycelium had grown well, it was ready for use. Other plant-derived fungi, *Fusarium graminearum* (…), were also cultured. Fusarium gramineae ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Corynebacterium tomatoense ( Corynespora cassicola ), Rhizoctonia solani ( Rhizoctonia solani Verticillium dahliae Verticillium wilt dahlias ), Watermelon Rhizoctonia solani ( Rhizoctonia solani (Cl) Fusarium oxysporum ( Fusarium oxysporum All were inoculated onto PDA medium and incubated in a constant temperature incubator at 25±0.1 ℃ for 3-6 days. Once the mycelium had grown well, they were ready for use.

[0067] Assay Method: In vitro inhibitory activity was determined using the mycelial growth rate method. The test compound and control agent were prepared into a 40 mM solution using DMSO (dimethyl sulfoxide). The calculated amount of solution was then mixed with PDA medium to prepare the test concentration. DMSO was used as a blank control. The medium was prepared into plates in 5 cm petri dishes, with three replicates for each treatment. After inoculating with 2 mm mycelial discs, the plates were placed in a constant temperature incubator at 27 ± 0.1 ℃. Once the colony diameter in the blank control plates reached 3–4 cm, the colony diameter of each treatment and the control was measured using the cross-sectional method.

[0068] Calculation of inhibition rate: Colony growth diameter (mm) = Colony measurement diameter (mm) - Cryopreservation diameter (mm); Mycelial growth inhibition rate (%) = [diameter of blank colony growth (mm) - diameter of colony growth on drug-containing medium (mm)] / diameter of control colony growth (mm) × 100.

[0069] Example 10: Inhibitory activity of the synthesized melatonin derivative against Penicillium italicum. Penicillium italicum: Prepare PDA medium, then add melatonin derivatives to the medium. The final concentration of melatonin added is (M1-M27: 200 µM). Place a 2 mm Penicillium italicum block in the middle of the medium and incubate at room temperature for 7 days. Each group has 3 replicates.

[0070] Observe the growth of pathogens, such as Figure 1 and Figure 2 As shown, when the substituents at different positions of melatonin are changed, its inhibitory activity against Penicillium italicum is significantly enhanced compared to melatonin alone.

[0071] Example 11: Inhibitory activity of melatonin derivatives against eight other plant pathogenic fungi Fusarium graminearum ( Fusarium gramineae Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Scrape 2 mm of mycelium from the *Rhizoctonia solani* block and place it in the middle of the culture medium. Incubate at room temperature for two days and calculate the inhibition rate. Each group has 3 replicates.

[0072] Botrytis cinerea ( Botrytis cinerea Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the culture medium. Incubate at room temperature in the dark for 4 days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0073] Sclerotium sclerotiorum ( Sclerotinia sclerotiorum Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm sized piece of mycelium and place it in the middle of the culture medium. Incubate at room temperature for two days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0074] Corynebacterium tomatoense ( Corynespora cassicola Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the culture medium. Incubate at room temperature for 3 days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0075] Rhizoctonia solani ( Rhizoctonia solani Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the culture medium. Incubate at room temperature for 1 day. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0076] Verticillium dahliae ( Verticillium dahliae Prepare PDA solid culture medium, then add compound M-20 to the culture medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the culture medium. Incubate at room temperature in the dark for 10 days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0077] Watermelon Rhizoctonia solani ( Rhizoctonia solani (Cl) Place PDA solid medium, then add compound M-20 to the medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the medium. Incubate at room temperature in the dark for 4 days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0078] Fusarium oxysporum ( Fusarium oxysporum Place PDA solid medium, then add compound M-20 to the medium. The final concentration of the added melatonin derivative is (50 µM). Take a 2 mm bacterial block and place it in the middle of the medium. Incubate at room temperature in the dark for 3 days. Measure the colony diameter using the cross-cross method and calculate the inhibition rate. Each group has 3 replicates.

[0079] Observe the growth of pathogens (e.g.) Figure 3 As shown in the figure, it can be seen that the melatonin derivative M-20 has a significant inhibitory effect on the above fungi.

[0080] The following shows the inhibitory effect of melatonin derivative M1-27 on Penicillium italicum: .

Claims

1. A melatonin derivative having the structure shown in formula (Ⅰ), Equation (I) in, R 1 Selected from H, methyl, and phenyl; R 2 Selected from phenyl, 4-fluorophenyl, 3-fluorophenyl, 2-fluorophenyl, 3,5-difluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 4-trifluoromethylphenyl, 4-chlorophenyl, 3,5-dichlorophenyl, 3,4-dichlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2-furanyl, 2-thienyl; R 3 Selected from H, halogen, methyl, methoxy, trifluoromethyl; R 3 It can be in any substitution position, either single or multiple substitution.

2. The compound according to claim 1, characterized in that, In formula (Ⅰ), each substituent is any one of the following: Compound M-1: R 1 For Ph, R 2 For Ph, R 3 For H; Compound M-2: R 1 CH3, R 2 For Ph, R 3 For H; Compound M-3: R 1 For H, R 2 For Ph, R 3 For H; Compound M-4: R 1 For H, R 2 It is 4-fluorophenyl, R 3 For H; Compound M-5: R 1 For H, R 2 It is 3-fluorophenyl, R 3 For H; Compound M-6: R 1 For H, R 2 It is 2-fluorophenyl, R 3 For H; Compound M-7: R 1 For H, R 2 It is 3,5-difluorophenyl, R 3 For H; Compound M-8: R 1 For H, R 2 It is 2,4-difluorophenyl, R 3 For H; Compound M-9: R 1 For H, R 2 It is 3,4-difluorophenyl, R 3 For H; Compound M-10: R 1 For H, R 2 It is 4-trifluoromethylphenyl, R 3 For H; Compound M-11: R 1 For H, R 2 It is 4-chlorophenyl, R 3 For H; Compound M-12: R 1 For H, R 2 It is 3,5-dichlorophenyl, R 3 For H; Compound M-13: R 1 For H, R 2 It is 3,4-dichlorophenyl, R 3 For H; Compound M-14: R 1 For H, R 2 It is 4-methylphenyl, R 3 For H; Compound M-15: R 1 For H, R 2 It is 4-methoxyphenyl, R 3 For H; Compound M-16: R 1 For H, R 2 It is 2-furanyl, R 3 For H; Compound M-17: R 1 For H, R 2 It is 2-thienyl, R 3 For H; Compound M-18: R 1 For H, R 2 It is 3-fluorophenyl, R 3 5-F; Compound M-19: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 6-F; Compound M-20: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5.6-2F; Compound M-21: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-CF3; Compound M-22: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 7-CH3; Compound M-23: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-CH3; Compound M-24: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-MeO; Compound M-25: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-Br; Compound M-26: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5-I; Compound M-27: R 1 For H, R 2 It is 3-fluorophenyl, R 3 It is 5,6-2Cl.

3. The method for preparing melatonin derivatives according to any one of claims 1 to 2, characterized in that, Includes the following steps: Step a: Mix raw material I, ammonium acetate, and nitromethane and react them to obtain intermediate II; Step b: Intermediate II, zinc acetate, and cyclohexane are mixed and then indole or an indole derivative is added to react and obtain intermediate III. Step c: Under N2 atmosphere protection, lithium aluminum hydride is slowly added to a solution containing intermediate III at -5~0 ℃, and the reaction is carried out at room temperature to obtain intermediate IV; In step d, triethylamine and intermediate IV were mixed in a solvent under a N2 atmosphere, and acid anhydride was slowly added at low temperature. The mixture was then raised to room temperature to give product V, as shown in the following reaction equation: 。 4. The preparation method according to claim 3, characterized in that, In step a, raw material I, ammonium acetate, and nitromethane are reacted at 80-100 °C for 1-2 h until the reaction is complete. This step also includes post-treatment, which involves removing unreacted nitromethane under reduced pressure after the reaction is complete, extracting and separating the liquid and combining the organic phases, drying and concentrating them for column chromatography to obtain the corresponding product II.

5. The preparation method according to claim 4, characterized in that, In step b, indole or an indole derivative is added to intermediate II, zinc acetate, and cyclohexane, and the mixture is reacted at 50-75 °C for 10-12 h until the reaction is complete. This step also includes a post-treatment, which involves removing the cyclohexane solvent under reduced pressure after the reaction is complete, extracting and separating the liquid and combining the organic phases, drying and concentrating the mixture, and then performing column chromatography to obtain the corresponding product III.

6. The preparation method according to claim 5, characterized in that, In step c, under N2 atmosphere protection, lithium aluminum hydride is slowly added to the tetrahydrofuran solution of intermediate III at 0 °C, and then slowly raised to room temperature for 6 h. This step also includes post-treatment, which involves quenching unreacted lithium aluminum hydride with water at 0 °C after the reaction is complete, followed by filtration with the addition of diatomaceous earth, combining the organic phases, drying and concentrating under reduced pressure to obtain an oily solid, and then purifying by column chromatography to obtain product IV.

7. The preparation method according to claim 6, characterized in that, In step d, triethylamine and dichloromethane solution of intermediate IV are slowly added to acid anhydride at low temperature under N2 atmosphere, and then the reaction is continued at room temperature for 10-12 h until the reaction is complete. This step also includes post-treatment, which involves adding a saturated aqueous solution of sodium bicarbonate to the system after the reaction is complete, extracting and separating the organic phases, combining them, drying and concentrating them, and performing column chromatography to obtain product V.

8. The melatonin derivative according to any one of claims 1 to 2 for the prevention and control of Penicillium citrus ( Penicillium italicum Applications on ) Or the melatonin derivatives according to any one of claims 1 to 2 for inhibiting Fusarium graminearum ( Fusarium graminearum ), Botrytis cinerea ( Botrytis cinerea ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Corynebacterium tomatoense ( Corynespora cassiicola ), Rhizoctonia solani ( Rhizoctonia solani Verticillium dahliae Verticillium dahliae ), Watermelon Rhizoctonia solani ( Rhizoctonia solani (Cl) Fusarium oxysporum ( Fusarium oxysporum Application of pesticides to any one or more plant diseases.

9. The application according to claim 8, characterized in that, The concentration of the melatonin derivative is 50-200 μM.

10. A pesticide fungicide, characterized in that, The pesticide fungicide includes any one or more combinations of the melatonin derivatives described in claim 1 or 2.

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