Coumarin schiff base derivatives, and preparation method and application thereof

Abstract

This invention discloses coumarin Schiff base derivatives. These derivatives are based on coumarin, and a series of coumarin derivatives with Schiff base structures are introduced through a reaction. These compounds exhibit good antifungal activity against *Botrytis cinerea*, *Alternaria alternata*, *Fusarium oxysporum*, and *Alternaria alternata*, opening up new avenues for the control of various plant fungal diseases and possessing broad development potential and promising application prospects. Furthermore, in vitro antioxidant experiments on these Schiff base compounds showed that they exhibit excellent scavenging abilities against DPPH radicals, ABTS radicals, and hydroxyl radicals, with antioxidant performance comparable to the positive control ascorbic acid, demonstrating their potential as novel antioxidants.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis, and more specifically, to coumarin Schiff base derivatives, their preparation methods, and applications. Background Technology

[0002] Plant fungal diseases not only severely restrict plant growth and development but also cause incalculable losses to plant yield and quality. To improve crop yield and quality, organic pesticides are commonly used in agriculture for disease control. However, long-term pesticide use has led to increased pathogen resistance, necessitating researchers to continuously explore new pesticide varieties with novel sites of action and mechanisms of action. Natural products possess excellent biological activity, but their extraction is difficult, resulting in challenges in large-scale extraction and inconsistent quality. In recent years, researchers have focused on using natural products as lead compounds, through artificial synthesis and structural modification, to develop novel, efficient, and environmentally friendly plant-derived fungicides.

[0003] Reactive oxygen species (ROS) are continuously produced by the body for normal oxygen use and are characterized by oxidative stress due to increased concentrations of oxidizing substances within cells. ROS include free radicals, such as superoxide anion radicals. Hydroxyl radicals (OH·), lipid peroxidation radicals (ROO·), hydrogen peroxide (H2O2), and singlet oxygen (OH·). 1 (O2). Studies have shown that excessive ROS attack many organs and directly induce oxidative damage to key biomolecules such as nucleic acids, lipids, proteins, enzymes, and sugars, leading to cardiovascular disease, cancer, and aging. Antioxidant active ingredients can scavenge free radicals and reduce oxidized substances, thus protecting the body. Finding and developing antioxidants that can scavenge oxygen free radicals has become an important research topic in the fields of biology, medicine, chemistry, and pharmaceutics.

[0004] Coumarin derivatives, based on benzopyranone, are secondary metabolites of plants and can be used as plant self-protective agents. Studies have reported that coumarins and their derivatives possess various pharmacological effects, including anti-inflammatory, antioxidant, anti-cancer, anti-tumor, and anticoagulant properties. These compounds have advantages such as small molecular weight, relatively simple synthesis, broad pharmacological activity, and low toxicity, attracting widespread attention from scholars both domestically and internationally.

[0005] Schiff bases are a class of organic compounds that possess imine or methylimine characteristic groups (-RC=N-). Compounds containing Schiff base structures often exhibit excellent antibacterial, antiviral, and other biological activities, and their synthesis methods are relatively simple and highly flexible.

[0006] However, the performance of existing coumarin derivatives still needs further improvement. Summary of the Invention

[0007] To address the problems in existing technologies, this invention proposes coumarin Schiff base derivatives, their preparation methods, and applications. The experimental research of this invention uses coumarin as the parent compound and introduces a series of coumarin derivatives with Schiff base structures through reactions. Antifungal experiments on compounds 3a-3o and 4a-4i show that these compounds exhibit good antifungal activity against *Botrytis cinerea*, *Alternaria alternata*, *Fusarium oxysporum*, and *Alternaria alternata*, expanding the scope of control for various plant fungal diseases and possessing broad development potential and promising application prospects. Furthermore, in vitro antioxidant experiments on these Schiff base compounds show that they exhibit excellent scavenging abilities against DPPH radicals, ABTS radicals, and hydroxyl radicals, with antioxidant performance comparable to the positive control ascorbic acid, demonstrating their potential as novel antioxidants.

[0008] One objective of this invention is to provide a coumarin Schiff base derivative, wherein the coumarin Schiff base derivative comprises the following structure:

[0009]

[0010] In structure [1] or structure [2],

[0011] R1 can be independently -OH, -H, or halogen;

[0012] R2 can be independently -H, -OH, halogen, or C1-C5 alkoxy groups;

[0013] R3 can be independently -H, C1-C5 alkoxy, -OH or halogen;

[0014] R4 can be independently -H, halogen, or -OH;

[0015] R5 is independently -H, halogen;

[0016] Furthermore, at least two of R1, R2, R3, R4, and R5 are not hydrogen; and none of the halogens in R1, R2, R3, R4, and R5 contain the element F.

[0017] In the coumarin Schiff base derivatives described in this invention, preferably,

[0018] The halogens in R1, R2, R3, R4, and R5 are each independently selected from -Cl and -Br;

[0019] Preferably,

[0020] R1 is -OH, -H, or -Cl; and / or,

[0021] R2 is an alkoxy group of -H, -OH, -Cl, -Br, or C1-C3; preferably -OCH3; and / or,

[0022] R3 is an alkoxy group of -H, -Br, -OH, -Cl, or C1-C3; preferably -OCH3; and / or,

[0023] R4 is -H, -Br, -Cl, -OH; and / or,

[0024] R5 can be -Br, -H, or -Cl.

[0025] In the coumarin Schiff base derivatives described in this invention, preferably, two or three of R1, R2, R3, R4, and R5 are not hydrogen.

[0026] Preferably, at least one of R1, R2, R3, R4, and R5 is -OH, and / or at least one of R1, R2, R3, R4, and R5 is a halogen; more preferably, each of the halogens is independently selected from -Cl and -Br.

[0027] In the coumarin Schiff base derivatives described in this invention, preferably,

[0028] R1 is -OH, and at least one of R2, R3, R4, and R5 is -OH, halogen, or -OCH3;

[0029] Preferably,

[0030] R1 is -OH, and only one of R2, R3, R4, and R5 is -OH; or one or two of R2, R3, R4, and R5 are halogens; or only one of R2, R3, R4, and R5 is -OCH3; and / or,

[0031] R1 is -Cl, and at least one of R2, R3, R4, and R5 is -OH or -Br; preferably, R1 is -Cl, and only one of R2, R3, R4, and R5 is -OH or -Br; and / or,

[0032] R1 is H, and at least two of R2, R3, R4, and R5 are halogens; the halogen is -Cl or -Br; preferably, R1 is H, and two of R2, R3, R4, and R5 are -Cl.

[0033] In the coumarin Schiff base derivatives of the present invention, preferably, the coumarin Schiff base derivatives are selected from the following compounds:

[0034]

[0035]

[0036] A second objective of this invention is to provide a method for preparing coumarin Schiff base derivatives as described in one objective of this invention, comprising the following steps:

[0037] Under the action of a catalyst, the substrate and the compound shown in formula [3] are reacted in a solvent to obtain the coumarin Schiff base derivatives;

[0038] The substrate is 3-aminocoumarin or 7-amino-4-methylcoumarin;

[0039] The compound represented by formula [3] is Wherein, R1, R2, R3, R4, and R5 in the formula [3] correspond to the same R1, R2, R3, R4, and R5 in the coumarin Schiff base derivatives described in one of the objectives of this invention.

[0040] In the preparation method of coumarin Schiff base derivatives described in this invention, preferably,

[0041] The catalyst is selected from at least one of acetic acid, acetic anhydride, phosphoric acid, or aluminum trichloride; and / or,

[0042] The solvent is selected from alcohol solvents, preferably at least one of methanol or ethanol; and / or,

[0043] The compound represented by formula [3] is selected from the following compounds:

[0044]

[0045]

[0046] Preferably,

[0047] The molar ratio of the substrate to the compound shown in formula [3] is 1:1 to 1.5; preferably, it is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or any range of parameters between the two values ​​mentioned above; and / or,

[0048] The molar volume ratio of the substrate to the solvent is 1 mmol: 10–50 mL; preferably, it is 1 mmol: 10 mL, 1 mmol: 20 mL, 1 mmol: 30 mL, 1 mmol: 40 mL, 1 mmol: 50 mL, or any range thereof; and / or,

[0049] The molar volume ratio of the substrate to the catalyst is 1 mmol: 0.05–0.1 mL; preferably, it is 1 mmol: 0.05 mL, 1 mmol: 0.06 mL, 1 mmol: 0.07 mL, 1 mmol: 0.08 mL, 1 mmol: 0.09 mL, 1 mmol: 0.1 mL, or any range thereof; and / or,

[0050] The reaction temperature is 70–85°C; preferably 70, 72, 74, 76, 78, 80, 82, 84, 85°C, or any parameter range between these two values; and / or,

[0051] The reaction time is 8 to 10 hours; preferably, it is 8, 9, or 10 hours or any parameter range between the two values ​​mentioned above.

[0052] In the technical solution of the present invention, the substrate solution and the compound solution shown in Formula [3] can be mixed, a catalyst can be added, and then the temperature can be raised to react to obtain the coumarin Schiff base derivative; or the substrate solution and the compound solution shown in Formula [3] can be mixed, and then the catalyst can be added and the temperature can be raised together to react to obtain the coumarin Schiff base derivative.

[0053] In the preparation method of coumarin Schiff base derivatives of the present invention, most preferably, the method includes:

[0054] (1) Dissolve 3-aminocoumarin or 7-amino-4-methylcoumarin in anhydrous methanol or anhydrous ethanol and use ultrasound to assist dissolution.

[0055] (2) Heat to 40°C, add benzaldehyde dissolved in methanol or ethanol dropwise to the mixture, and add 1-2 drops of glacial acetic acid or acetic anhydride for catalysis;

[0056] (3) After the addition is complete, the temperature is raised to 79°C and the reaction is carried out for 8 hours. After the reaction is completed, the suspension is collected by filtration and washed with methanol or ethanol. The resulting solid is the purified compound 3a-3o or 4a-4i.

[0057] The third objective of this invention is to provide the application of coumarin Schiff base derivatives as described in the first objective of this invention or the coumarin Schiff base derivatives prepared by the method described in the second objective of this invention in the preparation of antibacterial and antioxidant drugs.

[0058] Preferably, when used for antibacterial purposes, the fungus is plant-based, and more preferably, it is used in antibacterial agents containing at least one of the following pathogens: Botrytis cinerea, Alternaria solanacea, Fusarium oxysporum, or Alternaria.

[0059] In the technical solution of this invention, the inventors used the growth rate method to determine the fungicidal effect of the 3-aminocoumarin or 7-amino-4-methylcoumarin derivative involved in this invention on *Botrytis cinerea*, which causes gray mold in strawberries and tomatoes; *Altemaria solani*, which causes early blight in tomatoes; *Fusarium oxysporum*, which causes crop wilt; and *Alternaria alternata*, which causes black mold in tobacco and tomatoes. The inhibitory effect on the four pathogens was particularly good at 200 μg / mL.

[0060] The fourth objective of this invention is to provide an antibacterial drug, including the coumarin Schiff base derivatives described in the first objective of this invention or the coumarin Schiff base derivatives prepared by the method described in the second objective of this invention.

[0061] The fifth objective of this invention is to provide an antioxidant drug, including the coumarin Schiff base derivatives described in the first objective of this invention or the coumarin Schiff base derivatives prepared by the method described in the second objective of this invention.

[0062] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values; these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the following, various technical solutions can, in principle, be combined with each other to obtain new technical solutions, which should also be considered as specifically disclosed herein.

[0063] Compared with the prior art, the present invention has at least the following advantages:

[0064] This invention introduces amino groups at the C-3 and C-7 positions of the coumarin heterocycle and synthesizes a series of 3-aminocoumarin and 7-amino-4-methylcoumarin derivatives with Schiff base structures through reaction. The coumarin Schiff base derivatives of this invention have simple structures, small molecular weights, and good biological activity, exhibiting varying degrees of inhibition against test bacteria, and possess the potential to become lead compounds for the development of novel pesticides. Furthermore, in vitro antioxidant experiments on these Schiff base compounds showed that they exhibit excellent scavenging abilities against DPPH radicals, ABTS radicals, and hydroxyl radicals, with antioxidant performance comparable to the positive control ascorbic acid, demonstrating their potential as novel antioxidants. Detailed Implementation

[0065] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0066] It should also be noted that the various specific technical features described in the following embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the various possible combinations will not be described separately in this invention.

[0067] Furthermore, various embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention. The resulting technical solutions are part of the original disclosure of this specification and also fall within the protection scope of the present invention.

[0068] Unless otherwise specified, the raw materials used in the examples and comparative examples are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0069] Example 1

[0070] Synthesized compound 3a 3-((2-bromo-6-hydroxybenzylmethyl)amino)-coumarin:

[0071]

[0072] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0073] 2. Heat to 40℃, add 0.3014 g (1.5 mmol) of 2-bromo-6-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0074] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2077 g of pure orange solid product, with a yield of 60.36% and a melting point of 206-209℃.

[0075] Example 2

[0076] Synthesized compound 3b 3-((2-hydroxy-4-methoxybenzyl)amino)-coumarin:

[0077]

[0078] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0079] 2. Heat to 40℃, add 0.2282g (1.5mmol) of 2-hydroxy-4-methoxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0080] 3. The reaction process was monitored by thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to yellowish-brown and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2267 g of pure yellowish-brown solid, with a yield of 76.77% and a melting point of 184-185℃.

[0081] Example 3

[0082] Compound 3C 3-((4-bromo-2-hydroxybenzylmethyl)amino)-coumarin:

[0083]

[0084] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0085] 2. Heat to 40℃, add 0.3015g (1.5mmol) of 4-bromo-2-hydroxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0086] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1464 g of pure orange solid product, with a yield of 42.53% and a melting point of 258 °C.

[0087] Example 4

[0088] Synthesized compound 3d 3-((2-chloro-6-hydroxybenzylmethyl)amino)-coumarin:

[0089]

[0090] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0091] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 2-chloro-6-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0092] 3. The reaction process was monitored by thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange-yellow and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2563 g of pure orange-yellow solid, with a yield of 85.52% and a melting point of 210℃.

[0093] Example 5

[0094] Synthesized compound 3e 3-((2-chloro-4-hydroxybenzyl)amino)-coumarin

[0095]

[0096] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0097] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 2-chloro-4-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0098] 3. The reaction progress was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange-yellow and a solid precipitated. The reaction was then stopped. After cooling to room temperature, the product was washed with methanol, filtered, and dried to obtain 0.1005 g of pure orange-yellow solid, with a yield of 33.53% and a melting point of 208℃.

[0099] Example 6

[0100] Synthesized compound 3f 3-((4-chloro-2-hydroxybenzylmethyl)amino)-coumarin:

[0101]

[0102] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0103] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 4-chloro-2-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0104] 3. The reaction process was monitored by thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange-yellow and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1415 g of pure orange-yellow solid, with a yield of 47.21% and a melting point of 230℃.

[0105] Example 7

[0106] Synthesized compound 3g 3-((2,3-dihydroxybenzyl)amino)-coumarin:

[0107]

[0108] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0109] 2. Heat to 40℃, add 0.2072 g (1.5 mmol) of 2,3-dihydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0110] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to brown and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1650 g of pure brown solid product, with a yield of 58.66% and a melting point of 231-234℃.

[0111] Example 8

[0112] Synthesized compound 3h 3-((2,4-dihydroxybenzyl)amino)-coumarin:

[0113]

[0114] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0115] 2. Heat to 40℃, add 0.2072 g (1.5 mmol) of 2,4-dihydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0116] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to brown and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1066 g of pure brown solid product, with a yield of 37.90% and a melting point of 211-213℃.

[0117] Example 9

[0118] Compound 3i 3-((2,5-dihydroxybenzyl)amino)-coumarin:

[0119]

[0120] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0121] 2. Heat to 40℃, add 0.2072 g (1.5 mmol) of 2,5-dihydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0122] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to brown and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2246 g of pure brown solid product, with a yield of 79.84% and a melting point of 244-248℃.

[0123] Example 10

[0124] Compound 3j 3-((3,5-dichlorobenzylmethyl)amino)-coumarin:

[0125]

[0126] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0127] 2. Heat to 40℃, add 0.2625g (1.5mmol) of 3,5-dichlorobenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0128] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange-red and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2220 g of pure orange-red solid product, with a yield of 69.45% and a melting point of 225-230℃.

[0129] Example 11

[0130] Compound 3k 3-((3,5-dichloro-2-hydroxybenzylmethyl)amino)-coumarin:

[0131]

[0132] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0133] 2. Heat to 40℃, add 0.2865g (1.5mmol) of 3,5-dichloro-2-hydroxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0134] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2720 g of pure orange solid product, with a yield of 81.39% and a melting point of 220-223℃.

[0135] Example 12

[0136] Compound 3l 3-((3,5-dibromo-2-hydroxybenzylmethyl)amino)-coumarin:

[0137]

[0138] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0139] 2. Heat to 40℃, add 0.4199g (1.5mmol) of 3,5-dibromo-2-hydroxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0140] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.3702 g of pure orange solid product, with a yield of 81.39% and a melting point of 220-223℃.

[0141] Example 13

[0142] Compound 3m 3-((2-hydroxy-3-methoxybenzyl)amino)-coumarin:

[0143]

[0144] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0145] 2. Heat to 40℃, add 0.2282 g (1.5 mmol) of 2-hydroxy-3-methoxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0146] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1776 g of pure orange solid product, with a yield of 60.14% and a melting point of 204-206℃.

[0147] Example 14

[0148] Compound 3n 3-((3-bromo-2-hydroxybenzylmethyl)amino)-coumarin:

[0149]

[0150] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0151] 2. Heat to 40℃, add 0.3015g (1.5mmol) of 3-bromo-2-hydroxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0152] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to pink and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2738 g of pure pink solid product, with a yield of 79.55% and a melting point of 230-233℃.

[0153] Example 15

[0154] Compound 3o 3-((4-bromo-2-chlorobenzyl)amino)-coumarin:

[0155]

[0156] 1. Dissolve 0.1620 g (1 mmol) of 3-aminocoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0157] 2. Heat to 40℃, add 0.3292g (1.5mmol) of 4-bromo-2-chlorobenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 3-aminocoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0158] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1006 g of pure orange solid product, with a yield of 19.23% and a melting point of 199-200℃.

[0159] Example 16

[0160] Compound 4a 7-((2-bromo-6-hydroxybenzyl)amino)-4-methyl-coumarin:

[0161]

[0162] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0163] 2. Heat to 40℃, add 0.3014 g (1.5 mmol) of 2-bromo-6-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0164] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange-red and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2193 g of pure orange-red solid product, with a yield of 61.22% and a melting point of 208℃.

[0165] Example 17

[0166] Compound 4b 7-((2-chloro-6-hydroxybenzyl)amino)-4-methyl-coumarin:

[0167]

[0168] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0169] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 2-chloro-6-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0170] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2004 g of pure orange solid product, with a yield of 63.88% and a melting point of 210℃.

[0171] Example 18

[0172] Compound 4c 7-((2-chloro-4-hydroxybenzyl)amino)-4-methyl-coumarin:

[0173]

[0174] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0175] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 2-chloro-4-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0176] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1593 g of pure orange solid product, with a yield of 50.87% and a melting point of 210℃.

[0177] Example 19

[0178] Compound 4d 7-((4-chloro-2-hydroxybenzyl)amino)-4-methyl-coumarin:

[0179]

[0180] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0181] 2. Heat to 40℃, add 0.2349 g (1.5 mmol) of 4-chloro-2-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0182] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2567 g of pure orange solid product, with a yield of 81.83% and a melting point of 228-230℃.

[0183] Example 20

[0184] Compound 4e 7-((2,5-dihydroxybenzyl)amino)-4-methyl-coumarin:

[0185]

[0186] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0187] 2. Heat to 40℃, add 0.2072 g (1.5 mmol) of 2,5-dihydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0188] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to brick red and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.1562 g of pure brick red solid product, with a yield of 52.90% and a melting point of 249℃.

[0189] Example 21

[0190] Compound 4f 7-((3,5-dichlorobenzylmethyl)amino)-4-methyl-coumarin:

[0191]

[0192] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0193] 2. Heat to 40℃, add 0.2625g (1.5mmol) of 3,5-dichlorobenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0194] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to yellow and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2540 g of pure yellow solid product, with a yield of 76.88% and a melting point of 248-250℃.

[0195] Example 22

[0196] Compound 4g 7-((3-methoxy-2-hydroxybenzyl)amino)-4-methyl-coumarin

[0197]

[0198] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0199] 2. Heat to 40℃, add 0.2282 g (1.5 mmol) of 3-methoxy-2-hydroxybenzaldehyde dissolved in 10 mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05 mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0200] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2305 g of pure orange solid product, with a yield of 74.55% and a melting point of 198-201℃.

[0201] Example 23

[0202] Compound 4h 7-((3-bromo-2-hydroxybenzyl)amino)-4-methyl-coumarin:

[0203]

[0204] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0205] 2. Heat to 40℃, add 0.3015g (1.5mmol) of 3-bromo-2-hydroxybenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0206] 3. The reaction process was monitored using thin-layer chromatography (TLC). During the reaction, the solution changed from colorless to pale yellow and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2020 g of pure pale yellow solid, with a yield of 56.39% and a melting point of 256-258℃.

[0207] Example 24

[0208] Compound 4i 7-((4-bromo-2-chlorobenzyl)amino)-4-methyl-coumarin:

[0209]

[0210] 1. Dissolve 0.1752 g (1 mmol) of 7-amino-4-methylcoumarin in 20 mL of anhydrous methanol using ultrasound assistance;

[0211] 2. Heat to 40℃, add 0.3292g (1.5mmol) of 4-bromo-2-chlorobenzaldehyde dissolved in 10mL of anhydrous methanol dropwise to a round-bottom flask containing 7-amino-4-methylcoumarin, then add 0.05mL of acetic acid, and stir and reflux at 79℃ for 8 hours.

[0212] 3. The reaction process was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to orange and a solid precipitated. The reaction was stopped, cooled to room temperature, and the product was washed with methanol, filtered, and dried to obtain 0.2280 g of pure orange solid product, with a yield of 60.54% and a melting point of 201℃.

[0213] Spectral data testing: Utilizing modern spectroscopic techniques, proton nuclear magnetic resonance (NMR) spectroscopy (H2N) 1 H NMR and carbon nuclear magnetic resonance (NMR) 13 The structure of the synthesized compound was characterized by CNMR.

[0214] 1. Compound 3a

[0215] 11H NMR (600 MHz, CDCl3): δ 9.88 (s, 1H), 7.71 (s, 1H), 7.60 - 7.53 (m, 2H), 7.39 (dd, J = 8.2, 1.0 Hz, 1H), 7.34 (td, J = 7.0, 1.8 Hz, 1H), 7.30 - 7.25 (m, 2H), 7.21 (t, J = 8.1 Hz, 1H).

[0216] 13 13C NMR (151 MHz, CDCl3) δ 197.79, 166.86, 163.30, 137.48, 134.45, 133.58, 131.65, 128.12, 124.90, 124.44, 123.80, 117.83, 117.41, 116.49, 116.09, 110.79.

[0217] 2. Compound 3b

[0218] 1 1H NMR (600 MHz, CDCl3) δ 13.25 (s, 1H), 9.17 (s, 1H), 7.46 (s, 1H), 7.37 (dd, J = 7.8, 1.6 Hz, 1H), 7.36 - 7.33 (m, 1H), 7.20 (d, J = 8.2 Hz, 1H), 6.35 - 6.32 (m, 1H), 3.69 (s, 3H).

[0219] 13 13C NMR (151 MHz, CDCl3) δ 194.32, 165.80, 158.08, 151.91, 134.54, 132.58, 130.93, 127.74, 124.76, 119.70, 116.31, 113.14, 107.54, 101.02, 55.46.

[0220] 3. Compound 3c

[0221] 1 1H NMR (600 MHz, CDCl3) δ 11.12 (s, 1H), 9.50 (s, 1H), 7.74 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 9.3 Hz, 1H), 7.32 - 7.26 (m, 2H), 7.23 - 7.18 (m, 2H), 6.71 (s, 1H).

[0222] 13C NMR(151MHz,CDCl3)δ195.94,166.51,162.08,135.23,134.68,134.27,131.89,128.31,126.79,125.18,123.69,123.02,121.26,120.87,116.65,111.02.

[0223] 4. Compound 3d

[0224] 1 H NMR(600MHz,CDCl3)δ9.89(s,1H),7.71(s,1H),7.57(dd,J=7.7,1.5Hz,1H),7.56 - 7.53(m,1H),7.42 - 7.37(m,1H),7.33(td,J=7.5,1.1Hz,1H),7.31 - 7.28(m,1H),7.28 - 7.26(m,1H),6.95(dt,J=7.9,0.9Hz,1H).

[0225] 13 C NMR(151MHz,CDCl3)δ195.47,164.35,163.36,157.73,152.38,149.06,137.40,134.34,133.60,131.71,128.19,126.63,124.98,121.00,120.38,117.13,116.73,116.29,110.86.

[0226] 5. Compound 3e

[0227] 1 H NMR(600MHz,CDCl3)δ7.71(s,1H),7.57(d,J=7.6Hz,1H),7.55(d,J=7.2Hz,1H),7.40 - 7.37(m,1H),7.33(td,J=7.5,1.1Hz,1H),7.31 - 7.28(m,1H),7.28 - 7.26(m,1H),6.95(s,1H),6.91(s,1H).

[0228] 13 C NMR(151MHz,CDCl3)δ195.81,164.69,137.74,134.68,133.94,132.06,128.53,126.97,125.42,124.97,121.34,120.72,117.47,117.07,116.51,111.21.

[0229] 6. Compound 3f

[0230] 1 H NMR (600MHz, CDCl3) δ11.17(s,1H),9.50(s,1H),7.73(s,1H),7.58(s,1H),7.56-7.54(m,1H),7.49(d,J=7.6 Hz,1H),7.41-7.37(m,1H),7.34(td,J=7.5,1.1Hz,1H),7.31-7.28(m,1H),7.21(ddd,J=7.6,6.0,2.5Hz,1H).

[0231] 13 C NMR (151MHz, CDCl3) δ195.92,166.57,162.43,135.33,132.07,128.51,127.00,1 25.43,125.38,124.98,121.02,120.36,118.35,118.02,116.85,116.54,111.23.

[0232] 7. Compound 3g

[0233] 1 H NMR (600MHz, CDCl3) δ11.09(s,1H),9.41(s,1H),7.71(s,1H),7.57(dd,J=13.5,7.9Hz,1H),7.39(d,J=8.3Hz,1H),7.34(t,J= 7.5Hz,1H),7.23-7.17(m,1H),7.16(d,J=7.8Hz,1H),7.05(dd,J=31.0,9.3Hz,1H),6.91(dt,J=48.4,7.8Hz,1H),6.71(s,1H).

[0234] 13 C NMR (151MHz, CDCl3) δ196.75,148.32,128.08,126.65,125.08,124.63,124.37,121.52,121.18,120.27,119.51,118.53,116.54,116.19,110.89.

[0235] 8. Compound 3h

[0236] 1H NMR (600MHz, DMSO) δ14.21(s,1H),10.04(s,1H),9.26(s,1H),7.98(d,J=2.5Hz,1H),7.76(d,J=7.8Hz,1H),7. 65(t,J=7.8Hz,1H),7.47(d,J=8.3Hz,1H),7.43(s,1H),7.42-7.37(m,2H),7.26(d,J=9.7Hz,1H),6.71(s,1H).

[0237] 13 C NMR (151MHz, DMSO) δ189.50,158.45,147.73,134.16,133.13,130.52,126.4 2,126.38,126.34,125.16,124.64,124.63,124.30,121.62,115.24,107.49.

[0238] 9. Compound 3i

[0239] 1 H NMR (600MHz, DMSO) δ11.76(s,1H),9.14(s,1H),9.08(s,1H),8.01(s,1H),7.72(s,1H),7.58(ddd,J=8.7,7.3,1.6Hz,1 H),7.43(d,J=8.3Hz,1H),7.37(td,J=7.5,1.1Hz,1H),7.04(d,J=3.0Hz,1H),6.90(dd,J=8.8,3.0Hz,1H),6.82(s,1H).

[0240] 13 C NMR (151MHz, DMSO) δ191.46,158.74,155.16,149.46,148.02,138.64,133.41,132.75,125.46,124.92,124.60,121.90,121.02,115.53,107.80.

[0241] 10. Compound 3j

[0242] 11H NMR (600 MHz, DMSO) δ 9.96 (s, 1H), 7.97 (s, 1H), 7.92 (s, 1H), 7.43 (s, 1H), 7.39 (s, 1H), 7.28 - 7.24 (m, 1H), 7.23 (dd, J = 7.2, 1.8 Hz, 1H), 7.20 (dd, J = 7.3, 1.7 Hz, 1H), 6.71 (s, 1H).

[0243] 13 13C NMR (151 MHz, DMSO) δ 188.94, 161.35, 159.96, 158.61, 147.89, 133.28, 130.35, 125.32, 124.78, 124.46, 121.77, 116.02, 115.39, 112.25, 107.67, 103.55.

[0244] 11. Compound 3k

[0245] 1 1H NMR (600 MHz, CDCl3) δ 13.89 (s, 1H), 9.58 (s, 1H), 7.80 (s, 1H), 7.59 (dt, J = 7.9, 3.5 Hz, 2H), 7.48 (d, J = 2.5 Hz, 1H), 7.42 - 7.33 (m, 3H).

[0246] 13 13C NMR (151 MHz, CDCl3) δ 195.18, 165.46, 157.68, 155.97, 152.48, 131.24, 130.86, 128.61, 125.31, 125.22, 124.77, 120.61, 119.42, 116.70, 116.32, 111.01.

[0247] 12. Compound 3l

[0248] 1 1H NMR (600 MHz, DMSO) δ 11.37 (s, 1H), 9.97 (s, 1H), 7.60 (s, 1H), 7.44 - 7.41 (m, 1H), 7.39 (dd, J = 1,6.8, 1.6 Hz, 1H), 7.28 - 7.25 (m, 1H), 7.24 - 7.21 (m, 1H), 7.19 (dd, J = 7.3, 5.7 Hz, 1H), 6.71 (s, 1H).

[0249] 13C NMR(151MHz,DMSO)δ188.74,158.73,148.02,138.42,135.39,133.95,133.4 1,130.76,128.94,125.45,124.92,124.60,121.90,116.08,115.53,107.78.

[0250] 13. Compound 3m

[0251] 1 H NMR(600MHz,DMSO)δ12.66(s,1H),9.21(s,1H),7.78-7.71(m,1H),7.60(s,1H), 7.40(td,J=7.3,1.8Hz,2H),7.26-7.24(m,2H),6.95-6.89(m,2H),3.84(s,3H).

[0252] 13 C NMR(151MHz,DMSO)δ165.13,160.33,154.38,153.69,153.50,152.44,150.24,1 26.84,122.45,119.82,118.92,118.45,117.85,117.16,114.02,108.95,18.60.

[0253] 14. Compound 3n

[0254] 1 H NMR (400MHz, CDCl3) δ14.05(s,1H),9.56(s,1H),7.76(d,J=8.8Hz,1H),7.64(d,J=7.9Hz,1 H),7.56(t,J=7.4Hz,2H),7.45-7.31(m,3H),6.95(t,J=7.8Hz,1H),6.86(t,J=7.8Hz,1H).

[0255] 13 C NMR (101MHz, CDCl3) δ195.84,166.13,157.49,152.04,139.82,136.72,135.8 3,132.30,131.77,130.20,128.14,124.88,119.95,119.21,116.29,110.90.

[0256] 15. Compound 3o

[0257] 11H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 9.52 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 13.4 Hz, 1H), 7.52 (dt, J = 17.1, 7.8 Hz, 1H), 7.41 - 7.27 (m, 1H), 6.70 (s, 1H), 6.35 (s, 1H).

[0258] 13 13C NMR (101 MHz, CDCl3) δ 137.73, 135.60, 133.75, 133.64, 133.12, 131.70, 131.35, 131.02, 130.81, 130.07, 128.47, 127.07, 125.24, 120.19, 116.91.

[0259] 16. Compound 4a

[0260] 1 1H NMR (600 MHz, CDCl3) δ 13.85 (s, 1H), 9.18 (s, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.26 - 7.25 (m, 1H), 7.25 - 7.22 (m, 1H), 6.99 (d, J = 8.4 Hz, 1H), 2.47 (d, J = 1.3 Hz, 3H).

[0261] 13 13C NMR (151 MHz, CDCl3) δ 164.56, 163.20, 160.45, 154.39, 151.81, 150.84, 134.59, 126.66, 125.66, 123.82, 118.88, 118.21, 117.50, 114.75, 109.11, 101.10, 18.49.

[0262] 17. Compound 4b

[0263] 1 1H NMR (600 MHz, DMSO) δ 13.93 (s, 1H), 9.24 (d, J = 1.5 Hz, 1H), 7.89 (dd, J = 8.4, 1.6 Hz, 1H), 7.59 (d, J = 2.1 Hz, 1H), 7.51 (dt, J = 6.1, 1.8 Hz, 1H), 7.50 - 7.48 (m, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.44 (s, 1H), 2.49 (s, 3H). <13 C NMR(151MHz,DMSO)δ163.06,162.69,160.21,154.33,153.40,150.77,136.25,1 35.42,127.04,120.85,119.15,118.90,117.14,116.01,114.50,109.57,18.60.

[0265] 18. Compound 4c

[0266] 1 H NMR(600MHz,DMSO)δ13.93(s,0H),7.89(dd,J=8.4,1.6Hz,1H),7.59(s,1H),7.53-7.4 7(m,2H),7.12(d,J=7.8Hz,1H),7.02(d,J=8.4Hz,1H),6.44(s,1H),2.55-2.47(m,3H).

[0267] 19. Compound 4d

[0268] 1 H NMR (600MHz, CDCl3) δ13.75(s,1H),9.21(s,1H),7.66(d,J=8.9Hz,1H),7.32(t,J=8.2Hz,1H),7.26(d,J=2.0Hz,2H),7. 27-7.23(m,2H),6.97(dd,J=7.9,1.1Hz,1H),6.95(dd,J=8.3,1.1Hz,1H),6.30(q,J=1.3Hz,1H),2.47(d,J=1.3Hz,3H).

[0269] 13 C NMR (151MHz, CDCl3) δ163.16,161.95,160.44,154.41,151.78,151.03,136.49,1 34.39,125.65,120.33,118.86,118.12,116.73,115.83,114.75,109.16,18.63.

[0270] 20. Compound 4e

[0271] 1H NMR(600MHz,DMSO)δ11.79(s,1H),9.15(s,1H),8.92(s,1H),7.82(s,1H),7.43(s,1H),7.38 (s,1H),7.09(s,1H),6.90(d,J=8.8Hz,1H),6.82(d,J=8.8Hz,1H),6.37(s,1H),2.45(s,3H).

[0272] 13 C NMR(151MHz,DMSO)δ164.77,159.98,154.03,153.34,153.15,152.08,149.89,1 26.49,122.09,119.47,118.56,118.10,117.50,116.81,113.67,108.60,18.24.

[0273] 21. Compound 4f

[0274] 1 H NMR (600MHz, CDCl3) δ8.61(s,1H),7.67(s,1H),7.53-7.49(m,1H),7.37(s,1H),7.24(d,J=7.6Hz,2H),6.32(s,1H),2.47(s,3H).

[0275] 13 C NMR (151MHz, CDCl3) δ194.97,162.35,155.84,133.51,131.03,130.15,125.83,119.86,118.16,115.03,108.80,101.12,18.51.

[0276] 22. Compound 4g

[0277] 1 H NMR (400MHz, CDCl3) δ13.23 (s, 1H), 8.72 (s, 1H), 7.70 (d, J = 8.3Hz, 1H), 7.34-7.25 (m, 2H), 7.1 1(ddd,J=12.2,7.9,1.5Hz,2H),6.98(t,J=7.9Hz,1H),6.33(s,1H),4.01(s,3H),2.52(s,3H).

[0278] 13C NMR (101MHz, CDCl3) δ196.61,164.44,160.59,154.39,151.95,151.45,151.24,148.46, 125.59,124.15,118.93,118.74,118.56,118.24,115.49,114.50,108.70,56.20,18.66.

[0279] 23. Compound 4h

[0280] 1 H NMR (400MHz, CDCl3) δ13.33(s,1H),11.11(s,1H),9.49(s,1H),7.68(s,1H),7.57 (s,3H),7.42-7.28(m,3H),7.22-6.94(m,5H),6.89(t,J=7.8Hz,1H),3.93(s,3H).

[0281] 13 C NMR (101MHz, CDCl3) δ196.61,167.32,157.77,152.11,151.50,148.34,134.64,1 31.49,128.12,124.87,124.72,119.50,119.15,118.81,116.36,115.43,56.19.

[0282] 24. Compound 4i

[0283] 1 H NMR (400MHz, CDCl3) δ8.84 (s, 1H), 8.12 (d, J = 8.4Hz, 1H), 7.68-7.59 (m, 2H), 7.52(d,J=9.5Hz,1H),7.15(d,J=8.2Hz,2H),6.26(s,1H),2.50-2.42(m,3H).

[0284] 13 C NMR (101MHz, CDCl3) δ160.83,157.70,154.65,154.37,152.11,137.04,132.73,1 31.63,130.73,129.81,126.69,125.45,118.27,117.75,114.33,108.90,18.74.

[0285] Antibacterial activity of coumarin Schiff base derivatives:

[0286] First, preliminary in vitro antibacterial activity tests were conducted on the 24 synthesized compounds. The test bacteria and experimental procedures are as follows:

[0287] 1. Test bacteria used in the experiment

[0288] Table 1

[0289]

[0290] The above four standard bacterial strains were used in this part of the experiments. All strains were provided by the Chemical Ecology Laboratory of the Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, and can also be purchased directly from Gary Chemical Network, Shanghai Boco Biotechnology Co., Ltd., etc.

[0291] 2. Preparation of PDA culture medium

[0292] (1) Weighing and cooking: Calculate the total amount of culture medium required based on the requirement of 10 mL of PDA medium per petri dish. Weigh a certain amount of peeled potatoes according to the calculation of adding 200 g of potatoes to every 1000 mL of distilled water. Cut the potatoes into small pieces and put them in a pot. Add 1000 mL of water and heat to a boil in the pot. Cook the potatoes until soft but not mushy. Filter while hot through 6-8 layers of gauze and discard the residue. Add water to the filtrate to 1000 mL.

[0293] (2) Heating to dissolve: Put the filtrate into a pot, add glucose (20g / 1000mL distilled water) and agar powder (18g / 1000mL distilled water), heat over low heat and stir constantly with a glass rod to prevent the agar powder from sticking to the bottom or overflowing. After the agar is completely dissolved, add water to the required amount.

[0294] (3) Dispensing and autoclaving: Dispense the prepared culture medium into 500ml Erlenmeyer flasks. It is advisable to dispense into the Erlenmeyer flasks no more than half full of their volume, seal them with sealing film, and autoclave them for later use.

[0295] 3. Solution preparation of synthesized coumarin Schiff base compounds

[0296] The compound solution was prepared to a concentration of 200 μg / mL by accurately weighing 24 mg of the compound into a centrifuge tube, adding 1200 μL of dimethyl sulfoxide (DMSO) using a pipette, and sonicating until completely dissolved. The compound solution was then sterilized under a UV sterilizer for 30 min.

[0297] 4. Experimental Procedure

[0298] Place 10 mL of PDA medium in a petri dish, add 100 μL of the compound solution, gently shake to mix, label, and cool horizontally (drug concentration: 200 μg / mL). Use 100 μL of dimethyl sulfoxide (DMSO) as a blank control. Perform three parallel experiments. Use commercially available drugs carbendazim and chlorothalonil as positive controls. Use a 0.7 cm diameter punch to collect vigorous mycelial cakes and punch concentric circles. Inoculate the center of fresh medium and invert the dish in a 25°C incubator. Measure the colony diameter after 96 hours using the cross-hatching method, calculate the average diameter, and determine the inhibitory rate of the compound.

[0299] Formula for calculating antibacterial rate: I = [(D0 - D] t [(D0-0.7)]×100%

[0300] (I: Mycelial growth inhibition rate, D0: Diameter of blank colony, D) t (Diameter of colonies after chemical treatment)

[0301] 5. Measurement Results

[0302] The inhibitory activity of the synthetic coumarin Schiff base derivatives against four common plant pathogens—Botrytis cinerea, Alternaria alternata, Fusarium oxysporum, and Alternaria alternata—was determined using the mycelial growth rate method described above. The experimental results are shown in Table 2.

[0303] Table 2

[0304]

[0305] Continued from Table 2

[0306]

[0307] Note: Higher values ​​in Table 2 indicate higher antibacterial activity, while negative values ​​indicate that the compound promotes fungal growth.

[0308] Based on the above results, we selected 3b, 3c, 3e, 3f, 3g, 3k, 3l, 3m, 3n, 3o, 4f, 4g, 4h, and 4i, which showed better antibacterial activity, for EC testing. 50 Testing. The test method was the colony diameter method using PDA medium. The compound test concentrations were: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. The comparative test concentrations of the commercially available drugs carbendazim and chlorothalonil were: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. The test data are shown in Table 3.

[0309] Table 3

[0310]

[0311] Continued from Table 3

[0312]

[0313] From EC 50 The test results show that, among these compounds, compound 3o exhibited inhibitory activity comparable to azoxystrobin against Alternaria alternata. However, compound 3f showed the best antibacterial activity against Alternaria solanacea, with an EC50 value of [missing information]. 50 The value was 16.07 μg / mL, far exceeding the efficacy of carbendazim and azoxystrobin, and comparable to that of chlorothalonil. Following 3f was 3k, whose EC50 value was... 50 The value was 47.27 μg / mL. For *Botrytis cinerea*, compound 4o showed relatively good inhibitory activity, with an EC50 value of 47.27 μg / mL. 50 The value was 84.61 μg / mL. For Fusarium oxysporum, compound 3n exhibited the best antibacterial activity, with an EC50 value of 84.61 μg / mL. 50 The value is 12.89 μg / mL. Following 3n is 3k, whose EC50 value is... 50 The value was 29.36 μg / mL. Furthermore, the EC50 values ​​for these 15 compounds were... 50 The value of R 2 All values ​​were greater than 0.9, indicating a good correlation between concentration and antibacterial effect. Overall, this series of compounds has a broad fungicidal spectrum and generally superior activity. The compounds are particularly effective in inhibiting early blight in tomatoes and wilt in crops, and may serve as lead compounds for the development of new pesticides.

[0314] In summary, the fungi targeted by this invention exhibit good inhibitory activity against four common basic plant fungi: *Botrytiscinerea* (causing gray mold in strawberries and tomatoes), *Altemaria solani* (causing early blight in tomatoes), *Fusarium oxysporum* (causing wilt in crops), and *Alternaria alternata* (causing black mold in tomatoes). These fungi can serve as lead compounds for the development of novel agricultural fungicides, used to treat or prevent fungal infections in plants. Therefore, the 3-aminocoumarin or 7-amino-4-methylcoumarin derivatives of this invention show significant efficacy in the preparation of antibacterial drugs, particularly antifungal drugs.

[0315] Antioxidant activity assay of coumarin Schiff base derivatives:

[0316] First, preliminary in vitro antioxidant activity tests were conducted on the 24 synthesized compounds. The experimental procedure is as follows:

[0317] 1. Solution preparation of synthesized coumarin Schiff base compounds

[0318] Using DMSO as solvent and 24 compounds as analytes, a stock solution with a concentration of 4 mg / mL was prepared and then successively diluted to 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL as test solutions, which were then refrigerated and protected from light for later use.

[0319] 2. Determination of DPPH free radical scavenging ability

[0320] (1) Preparation of DPPH test solution

[0321] Dissolve 1 mg of DPPH solid in 24 mL of anhydrous ethanol and sonicate until fully dissolved. Take 1 mL of the DPPH solution prepared in the above steps and add 0.5 mL of anhydrous ethanol to prepare a 0.1 mmol / L DPPH solution with an absorbance between 0.6 and 1.0. Store in the dark under cold conditions. If the absorbance is too high, add more solvent; if the absorbance is too low, add more DPPH or the original solution.

[0322] (2) Experimental methods

[0323] Add 1 mL of DMSO solution and 1 mL of DPPH solution to the same centrifuge tube, mix well, and let stand at room temperature for 30 minutes. Take 200 μL of the mixture and measure its absorbance at 519 nm. Repeat this process three times and take the average absorbance as A0. Similarly, add 1 mL of DPPH solution and 1 mL of the sample solution to the same centrifuge tube, mix thoroughly, and take 200 μL of the mixture to measure its absorbance at 519 nm. Repeat this process three times and take the average absorbance as A0. t .

[0324] Free radical scavenging formula: I = [(A0 - A t ) / A0]×100%

[0325] (I: DPPH free radical scavenging rate, A0: absorbance of the control group, A) t (sample group absorbance)

[0326] 3. Determination of ABTS· free radical scavenging ability

[0327] (1) ABTS· + Preparation of free radical working solution

[0328] Take 3 mg of ABTS diammonium salt in a centrifuge tube, add 0.735 mL of distilled water, and prepare a 7.4 mmol / L ABTS diammonium salt stock solution. Take 1 mg of K₂S₂O₈ solid, add 1.43 mL of distilled water, and prepare a 2.6 mmol / L K₂S₂O₈ stock solution. Refrigerate and protect from light for later use. Mix 0.2 mL of ABTS diammonium salt stock solution and 0.2 mL of K₂S₂O₈ stock solution thoroughly and react at room temperature in the dark for 12-14 hours. Dilute the mixture 30 times with PBS buffer (pH 7.4) until the absorbance at 734 nm is 0.700 ± 0.020. This solution is ABTS· + Free radical working solution.

[0329] (2) Experimental methods

[0330] Add 1 mL of DMSO solution and 1 mL of ABTS· + Add the free radical working solution to the same centrifuge tube, shake well, and let stand at room temperature for 30 minutes. Take 200 μL of the mixture and measure its absorbance at 734 nm. Repeat three times and take the average absorbance as A0. Similarly, add 1 mL of ABTS· + After mixing the free radical working solution and 1 mL of the sample solution in the same centrifuge tube, take 200 μL of the mixture and measure its absorbance at 734 nm. Repeat the process three times and take the average absorbance as A. t .

[0331] Free radical scavenging formula: I = [(A0 - A t ) / A0]×100%

[0332] (I: ABTS free radical scavenging rate, A0: absorbance of the control group, A) t (sample group absorbance)

[0333] 4. Determination of hydroxyl radical scavenging ability

[0334] (1) Solution preparation

[0335] Weigh 5g of ferrous sulfate, dissolve it in deionized water, and bring the volume to 500mL to prepare a ferrous sulfate solution with a concentration of 9mmol / L. Weigh 1.133g of 30% H₂O₂ solution, dissolve it in deionized water, and bring the volume to 1L to prepare a H₂O₂ solution with a concentration of 10mmol / L. Weigh 1.342g of salicylic acid, dissolve it in deionized water, and bring the volume to 1L to prepare a salicylic acid solution with a concentration of 9mmol / L. All prepared solutions should be refrigerated and protected from light for later use.

[0336] (2) Test method

[0337] Add 1 mL of deionized water, 1 mL of ferrous sulfate solution, and 1 mL of hydrogen peroxide solution to the same centrifuge tube and mix thoroughly. React at 37°C for 10 min. Then add 1 mL of salicylic acid solution to the centrifuge tube and react at 37°C for 30 min. Take 200 μL of the mixture and measure its absorbance at 510 nm. Repeat this process three times and take the average absorbance as A0. Similarly, add 1 mL of the test solution, 1 mL of ferrous sulfate solution, and 1 mL of hydrogen peroxide solution to the same centrifuge tube and mix thoroughly. React at 37°C for 10 min. Then add 1 mL of salicylic acid solution to the centrifuge tube and react at 37°C for 30 min. Take 200 μL of the mixture and measure its absorbance at 510 nm. Repeat this process three times and take the average absorbance as A0. t .

[0338] Free radical scavenging formula: I = [(A0 - A t ) / A0]×100%

[0339] (I: Hydroxyl radical scavenging rate, A0: Absorbance of control group, A) t (sample group absorbance)

[0340] 5. Measurement Results

[0341] By half-inhibition concentration IC 50 To evaluate the scavenging abilities of these 24 compounds against DPPH radicals, ABTS· radicals, and hydroxyl radicals, IC50 was used. 50 The smaller the value, the stronger the antioxidant activity of the corresponding compound. The corresponding IC50 value is calculated using a regression equation. 50 The values ​​and calculation results are shown in Table 4:

[0342] Table 4

[0343]

[0344] Continued from Table 4

[0345]

[0346] From IC 50 The test results show that compounds 3a-3o and 4a-4i have scavenging effects on all three types of free radicals. In terms of scavenging DPPH free radicals, compounds 3i, 3k, 3l, 3o, and 4e exhibit excellent DPPH free radical scavenging capabilities, with IC50 values ​​of [missing value]. 50 The values ​​were superior to those of the positive control ascorbic acid, especially the scavenging ability of compounds 4e and 3i against DPPH free radicals, IC50 values. 50 The values ​​reached 0.38 mg / mL and 0.49 mg / mL, respectively, which are the IC50 values ​​for ascorbic acid. 50The values ​​were 4 times and 3 times higher than those of their parent compounds, and 14 times and 9 times higher, respectively. Regarding ABTS· radical scavenging, almost all compounds showed ABTS· radical scavenging abilities close to those of the positive control ascorbic acid, demonstrating excellent free radical scavenging effects. Compounds 3m and 3j showed superior ABTS· radical scavenging abilities compared to ascorbic acid, reaching 1.09 mg / mL and 1.15 mg / mL, respectively. In terms of hydroxyl radical scavenging ability, compound 4a showed the best hydroxyl radical scavenging ability, but still did not reach the hydroxyl radical scavenging effect of the positive control ascorbic acid. Overall, this series of derivatives exhibits broad-spectrum free radical scavenging effects, particularly prominent in scavenging DPPH and ABTS· radicals, making them a class of lead compounds with broad antioxidant activity.

[0347] Note: In this invention, carbendazim, chlorothalonil, and azoxystrobin in Tables 2 and 3, and ascorbic acid in Table 4 are all used as positive controls. Since the substances in the above positive controls have significant structural differences from the coumarin Schiff base derivatives of this invention, the results of the positive controls are only used to prove the feasibility of the test method of this invention and the reliability of the experimental results. It does not mean that the results of some coumarin Schiff base derivatives of this invention are not as good as the results of the positive controls, but that the antibacterial and antioxidant properties of the coumarin Schiff base derivatives of this invention are poor.

[0348] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

[0349] All publications, patent applications, patents, and other references mentioned in this specification are incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. In case of conflict, the definitions in this specification shall prevail.

[0350] When this specification uses the prefixes “known to those skilled in the art,” “prior art,” or similar terms to derive materials, substances, methods, steps, apparatus, or components, the objects derived from such prefixes cover those commonly used in the art at the time of this application’s filing, but also include those that are not currently commonly used but will become generally recognized in the art as suitable for similar purposes.

[0351] In the context of this specification, except where expressly stated otherwise, any matters or issues not mentioned shall apply directly to those known in the art without any modification.

Claims

1. A coumarin Schiff base derivative, characterized in that, The coumarin Schiff base derivatives are compounds with the structure shown in [2]: Structure [2]; Structure [2] is selected from the following compounds: , , , , ; Alternatively, coumarin Schiff base derivatives are selected from the following compounds: 、 。 2. A method for preparing the coumarin Schiff base derivative as described in claim 1, characterized in that, The preparation method of the compound shown in structure [2] includes the following steps: Under the action of a catalyst, the substrate and the compound shown in formula [3] are reacted in a solvent to obtain the coumarin Schiff base derivative; The substrate is 7-amino-4-methylcoumarin; The compound represented by formula [3] is In this context, R1, R2, R3, R4, and R5 in the formula [3] correspond to the same R1, R2, R3, R4, and R5 in the coumarin Schiff base derivatives described in claim 1 as in structure [2].

3. The method for preparing coumarin Schiff base derivatives according to claim 2, characterized in that: The catalyst is selected from at least one of acetic acid, acetic anhydride, or phosphoric acid; and / or, The solvent is selected from alcohol solvents; and / or, The compound represented by formula [3] is selected from the following compounds: 、 、 、 、 。 4. The method for preparing coumarin Schiff base derivatives according to claim 3, characterized in that: The solvent is selected from at least one of methanol or ethanol; The molar ratio of the substrate to the compound shown in formula [3] is 1:1 to 1.5; and / or, The molar volume ratio of the substrate to the solvent is 1 mmol: 10–50 mL; and / or, The molar volume ratio of the substrate to the catalyst is 1 mmol: 0.05–0.1 mL; and / or, The reaction temperature is 70~85℃; and / or, The reaction time is 8 to 10 hours.

5. The application of a coumarin Schiff alkaloid derivative as described in claim 1 in the preparation of antifungal pesticides and antioxidant drugs; the fungus being Fusarium oxysporum.

6. A pesticide against plant fungi, characterized in that, Includes the coumarin Schiff base derivatives as described in claim 1.

7. An antioxidant drug, characterized in that, Includes the coumarin Schiff base derivatives as described in claim 1.

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