Coumarin compounds, methods of making and using the same

Abstract

The application discloses a coumarin derivative, a preparation method and application thereof, and has a structure as shown in formula (I) and / or (II). The method for preparing the coumarin derivative is simple and efficient, and the application is that the prepared coumarin derivative has good in-vitro inhibitory activity on four common plant pathogenic fungi of Botrytis cinerea, Fusarium oxysporum, Alternaria alternata and Alternaria solani. The compound has good application in a plant-derived fungus inhibitor, is more environmentally friendly, and meets the green chemistry concept.

Description

Technical Field

[0001] This invention relates to the technical field of chemical pharmaceuticals containing coumarin derivatives, and more specifically, to coumarin compounds, their preparation methods, and applications. Background Technology

[0002] Coumarin (1,2-benzopyranone) is one of the simplest classes of natural phenolic compounds, composed of a benzene ring and an α-pyranone ring. Studies have found that bioactive coumarin compounds isolated from natural products all have oxygen-containing substituents at the C-7 position of their parent ring structure. Therefore, coumarin and 7-hydroxycoumarin can both be considered the parent compounds of coumarin, and they mostly possess a wide range of biological activities, including anti-HIV, anti-tumor, anti-inflammatory, antioxidant, and antibacterial effects.

[0003] Heterocyclic structures like benzopyranones, due to their high efficiency, low toxicity, and diverse potential substituents, possess the potential for bioactivity similar to natural products, synthetic drugs, and pesticides. Modifying the C-7 position of methylcoumarin, by altering only the methyl side chain and linking its substituents to bromoalkanes, alkenes, or other substituents, can enhance the compound's lipophilicity and anti-inflammatory activity, inhibiting proteolytic inflammation to some extent—a finding confirmed in numerous coumarin synthesis bioactivity assays. With the deepening understanding of the antibacterial mechanisms of coumarins, finding candidates with lower toxicity and better efficacy, and providing novel plant-derived pesticides for the control of plant diseases and pests, has become a focus of attention. Current technologies have not yet disclosed highly efficient antibacterial agents for the synthesis of novel coumarin compounds. Summary of the Invention

[0004] To address the problems in existing technologies, this invention proposes coumarin compounds, their preparation methods, and applications. The coumarin compounds of this invention utilize C-8 methylcoumarin as the parent compound, substituting compounds at the C-7 and C-4 positions to prepare a variety of easily obtainable compounds. These compounds were then screened for activity against four common plant pathogenic fungi: *Botrytis cinerea*, *Alternaria solani*, *Fusarium oxysporum*, and *Alternaria alternata*. The mycelial growth rate method was used to determine the activity of the obtained compounds, revealing significant antibacterial activity. Furthermore, the coumarin compounds of this invention have low half-maximal effective concentrations (EC50) values, indicating lower toxicity. Therefore, the coumarin compounds of this invention possess the advantages of high efficiency and low toxicity in antibacterial activity, and have excellent application potential.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] One object of the present invention is to provide a coumarin compound selected from compounds represented by formula (I) and / or formula (II):

[0007]

[0008] The R1 is selected from H, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkenyl, substituted or unsubstituted C1-C20 alkyl carbonyl, heterocyclic carbonyl, C1-C20 alkoxydicarbonyl, and C1-C20 alkyl-substituted aromatic group.

[0009]

[0010] The R2 is selected from H, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkenyl, substituted or unsubstituted C1-C20 alkyl carbonyl, heterocyclic carbonyl, and C1-C20 alkoxy dicarbonyl.

[0011] In a preferred embodiment,

[0012] The R1 is selected from substituted or unsubstituted C1-C10 alkyl groups, substituted or unsubstituted C1-C10 alkenyl groups, substituted or unsubstituted C1-C10 alkyl carbonyl groups, oxygen-containing heterocyclic carbonyl groups or nitrogen-containing heterocyclic carbonyl groups, C1-C10 alkoxydicarbonyl groups, and C1-C10 alkyl-substituted aromatic groups; and / or,

[0013] The R2 is selected from substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkenyl, substituted or unsubstituted C1-C10 alkyl carbonyl, nitrogen-containing heterocyclic carbonyl, and C1-C10 alkoxydicarbonyl.

[0014] In a preferred embodiment,

[0015] The R1 is selected from substituted C1-C10 straight-chain alkyl or unsubstituted C1-C10 branched alkyl, substituted C1-C10 straight-chain alkenyl or unsubstituted C1-C10 branched alkenyl, substituted C1-C10 straight-chain alkyl carbonyl or unsubstituted C1-C10 branched alkyl carbonyl, oxygen-containing monocyclic heterocyclic carbonyl or nitrogen-containing monocyclic heterocyclic carbonyl, C1-C5 alkoxydicarbonyl, C1-C5 alkyl-substituted aromatic group; and / or,

[0016] The R2 is selected from substituted C1-C10 straight-chain alkyl or unsubstituted C1-C10 branched alkyl, substituted C1-C10 straight-chain alkenyl or unsubstituted C1-C10 branched alkenyl, substituted C1-C10 straight-chain alkyl carbonyl or unsubstituted C1-C10 branched alkyl carbonyl, nitrogen-containing monocyclic heterocyclic carbonyl, and C1-C5 alkoxydicarbonyl.

[0017] In a preferred embodiment,

[0018] In R1 or R2, the substituents in the substituted alkyl groups may be the same or different, and each is independently selected from halogens, nitrogen-containing heterocycles, preferably nitrogen-containing six-membered heterocycles; and / or,

[0019] The substituents in the substituted alkenyl group may be the same or different, and each is independently selected from halogens; and / or,

[0020] The substituents in the substituted alkyl carbonyl groups may be the same or different, and each may be independently selected from halogens or hydroxyl groups; and / or,

[0021] The number of branches in the unsubstituted alkyl group is 1; and / or,

[0022] The number of branches in an unsubstituted alkenyl group is 1; and / or,

[0023] The number of branches in the branched alkyl group of the unsubstituted alkyl carbonyl group is 1; and / or,

[0024] The alkyl group in the alkoxydicarbonyl group is a straight chain; and / or,

[0025] The heterocyclic group in the carbonyl group of R1 is an oxygen-containing aromatic heterocyclic group or a nitrogen-containing six-membered heterocyclic hydrocarbon group; and / or...

[0026] The alkyl group in the alkyl-substituted aromatic group of R1 is a straight chain; and / or,

[0027] In R2, the heterocyclic group in the heterocyclic carbonyl group is a nitrogen-containing six-membered heterocyclic hydrocarbon group.

[0028] In a preferred embodiment,

[0029] In R1 or R2, the halogen is selected from bromine; and / or,

[0030] The nitrogen-containing six-membered heterocyclic hydrocarbon group is an azacyclohexyl group; and / or...

[0031] The branched chain in the unsubstituted alkyl group is methyl; and / or,

[0032] The branched chain in the unsubstituted alkenyl group is methyl; and / or,

[0033] The branched alkyl group in the unsubstituted alkyl carbonyl group is methyl; and / or,

[0034] The alkenyl group is a monoalkenyl group; and / or,

[0035] The alkyl group in the alkoxydicarbonyl group is methyl, ethyl; and / or,

[0036] In R1, the oxygen-containing aromatic heterocyclic carbonyl group is selected from furan; and / or,

[0037] The aromatic group in an alkyl-substituted aromatic group is phenyl; the alkyl group in an alkyl-substituted aromatic group is methyl or ethyl.

[0038] In a preferred embodiment, the structure of R1 is as follows:

[0039]

[0040] And / or,

[0041] The structure of R2 is as follows:

[0042]

[0043] A second objective of this invention is to provide a method for preparing coumarin compounds, preferably for preparing the coumarin compounds described in one objective of this invention, comprising the following steps:

[0044] The preparation of the compound represented by formula (I) includes the following method:

[0045] Method 1: The starting materials, including 7-hydroxy-4-amino-8-methylcoumarin, an acid-binding agent, a phase transfer catalyst, and R1-X, are reacted in a solvent to produce the compound shown in formula (I); wherein X is a halogen, and R1 is selected from substituted or unsubstituted C1-C20 alkyl groups, substituted or unsubstituted C1-C20 alkenyl groups, and C1-C20 alkyl-substituted aromatic groups;

[0046] Method 2: The starting materials, including 7-hydroxy-4-amino-8-methylcoumarin and R1-X, undergo a substitution reaction in a solvent. Optionally, an alkylamine is added to undergo an acylation reaction to generate the compound shown in formula (I); wherein X is a halogen, and R1 is selected from substituted or unsubstituted C1-C20 alkyl carbonyl, heterocyclic carbonyl, or C1-C20 alkoxy dicarbonyl.

[0047] The preparation of the compound represented by formula (II) includes the following methods:

[0048] Method 3: The raw materials, including the compound shown in formula (I) and a dialkyl ketone, are reacted in concentrated acid to produce the compound shown in formula (II).

[0049] The preparation method described above in this invention also includes a post-processing step. The post-processing can employ conventional methods, and preferably includes the following:

[0050] (1) After the reaction is complete, a large amount of ice-cold distilled water is added for quenching and refrigeration. After filtration, the target product is obtained.

[0051] (2) Wash the solid with ethyl acetate, extract ethyl acetate three times with saturated sodium chloride solution, collect the organic phase, evaporate the organic phase to dryness by rotary evaporation, and separate the target product by column chromatography.

[0052] (3) Adjust the pH of the filtrate with 30% and 10% NaOH solutions until solids precipitate out. Collect the solids by filtration and separate them by column chromatography to obtain the target product.

[0053] The specific post-processing method can be selected according to the conventional methods used for the reaction system.

[0054] In the preparation method described in this invention, preferably,

[0055] In Method 1,

[0056] The acid-binding agent is selected from at least one of potassium carbonate or sodium carbonate; and / or,

[0057] The phase transfer catalyst is selected from at least one of TBAB and TBAI; and / or,

[0058] In R1-X, X is selected from bromine; and / or,

[0059] The solvent is selected from at least one of acetone, acetonitrile, or anhydrous DMF; and / or,

[0060] The reaction temperature is 50-60℃, preferably 52-56℃; and / or,

[0061] The reaction time is 10-40 h, preferably 20-32 h; and / or,

[0062] The molar ratio of 7-hydroxy-4-amino-8-methylcoumarin to the acid-binding agent is 1:1-6, preferably 1:2-4; and / or,

[0063] The molar ratio of 7-hydroxy-4-amino-8-methylcoumarin to the phase transfer catalyst is 1:0.1-3, preferably 1:0.5-2; and / or, the molar ratio of 7-hydroxy-4-amino-8-methylcoumarin to R1-X is 1:1-8, preferably 1:2-6; and / or,

[0064] The ratio of 7-hydroxy-4-amino-8-methylcoumarin to solvent is 1 mmol: 10-30 mL;

[0065] In method two,

[0066] In R1-X, X is selected from bromine; and / or,

[0067] The solvent is selected from at least one of acetone, acetonitrile, or anhydrous DMF; and / or,

[0068] Alkylamines are selected from triethylamine; and / or,

[0069] The acylation reaction temperature is 60-75℃, preferably 65-70℃; and / or

[0070] The temperature for the substitution reaction is 50-60°C, preferably 52-56°C; and / or,

[0071] The acylation reaction takes 5-8 hours, preferably 6.5-7 hours; and / or,

[0072] The substitution reaction takes 10-40 hours, preferably 20-32 hours; and / or,

[0073] The molar ratio of 7-hydroxy-4-amino-8-methylcoumarin to R1-X is 1:5-20, preferably 1:10-15; and / or,

[0074] The ratio of 7-hydroxy-4-amino-8-methylcoumarin to solvent is 1 mmol: 3-10 mL; and / or,

[0075] The ratio of 7-hydroxy-4-amino-8-methylcoumarin to triethylamine is 1 mmol: 0.2-2 mL;

[0076] In method three,

[0077] In the compound represented by formula (I), R1 is selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkenyl, substituted or unsubstituted C1-C20 alkyl carbonyl, heterocyclic carbonyl, and C1-C20 alkoxy dicarbonyl.

[0078] The dialkyl ketone is selected from 2,4-pentanedione; and / or,

[0079] The concentrated acid is selected from concentrated sulfuric acid; preferably, the concentrated sulfuric acid has a mass concentration of 70%-80%; and / or,

[0080] The reaction temperature is 100-135℃, preferably 120-130℃; and / or,

[0081] The reaction time is 3-6 hours, preferably 4-5 hours; and / or,

[0082] The molar ratio of the compound shown in formula (I) to the dialkyl ketone is 1:1-8, preferably 1:1-4; and / or,

[0083] The ratio of the compound shown in formula (I) to concentrated acid is 1 mmol: 0.3-2 mL.

[0084] More preferably, the present invention can first prepare 7-hydroxy-4-amino-8-methylcoumarin by step i in the following formula, and then prepare compound (I) by step ii (i.e., method one) or step iii (i.e., method two); and then prepare compound (II) by step IV (i.e., method three) of formula (I).

[0085]

[0086] In this invention, 7-hydroxy-4-amino-8-methylcoumarin can be prepared by other existing methods, or it can be synthesized by the following method: under the protection of an inert gas, raw materials including 2-methylresorcinol, cyanoacetic acid, and zinc chloride are reacted in a solvent under the condition of continuous HCl gas flow to produce 7-hydroxy-4-amino-8-methylcoumarin.

[0087] More preferably,

[0088] The molar ratio of 2-methylresorcinol to cyanoacetic acid is 1:0.5-3, preferably 1:1-2; and / or,

[0089] The molar ratio of 2-methylresorcinol to zinc chloride is 1:0.1-2, preferably 1:0.3-1; and / or,

[0090] The ratio of 2-methylresorcinol to solvent is 1 mmol: 20-100 mL; and / or,

[0091] The reaction temperature is room temperature; and / or,

[0092] The reaction time is 2-10 hours, preferably 3-5 hours.

[0093] A third objective of this invention is to provide the application of coumarin compounds described in one objective of this invention or prepared by the preparation method described in another objective of this invention in antibacterial applications, preferably in antifungal applications or as lead compounds for antibacterial agents, and more preferably in antifungal applications.

[0094] A fourth objective of this invention is to provide an antibacterial agent comprising the coumarin compounds described in one objective of this invention or the coumarin compounds prepared by the preparation method described in another objective of this invention.

[0095] 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.

[0096] Compared with the prior art, the compounds of the present invention have the following advantages:

[0097] (1) The coumarin compounds of the present invention have good in vitro antibacterial activity and strong antibacterial effects against Botrytis cinerea, Alternaria solanacea, Fusarium oxysporum and Alternaria solani. They can be used as antibacterial agents or antibacterial lead compounds.

[0098] (2) Existing coumarins are obtained from natural products, which is difficult to separate. The coumarin compounds of the present invention are synthesized by chemical methods. Moreover, the present invention has a wide variety of compounds and the synthesis method is simple, more environmentally friendly, and in line with the concept of green chemistry.

[0099] (3) The coumarin compounds of the present invention have low half-maximum effective concentration (EC50) values, corresponding to low toxicity and high relative inhibition rate. They have the advantages of high efficiency and low toxicity in antibacterial applications and can be used as antibacterial agents with excellent antibacterial effects. In particular, the coumarin compounds of the present invention have good applications in antibacterial agents for plant-derived fungi. Detailed Implementation

[0100] The present invention will now be described in detail with reference to specific examples and 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] Example 1

[0105] Synthesis of 7-hydroxy-4-amino-8-methylcoumarin

[0106]

[0107] All operations were performed under vacuum and nitrogen protection. HCl gas was continuously introduced. Preparation of HCl gas: A large amount of sodium chloride was dissolved in 400 mL of hydrochloric acid, and then sulfuric acid was added dropwise at room temperature. 18.61 g (0.15 mol) of 2-methylresorcinol, 12.75 g (0.15 mol) of cyanoacetic acid, and 11.06 g (0.08 mol) of zinc chloride were dissolved in 20 mL of diethyl ether and stirred at room temperature for 5 h. The solution changed from colorless to deep orange, then to pink with a white solid precipitating out. After the reaction was complete, a large amount of ice-cold distilled water was added for quenching and refrigeration. After filtration, a white powdery solid 2 was obtained.

[0108] 1 H NMR (400MHz, CD3OD_SPE) δ7.60(s,1H),6.82(s,1H),5.25(s,1H),2.26(s,3H).

[0109] Example 2

[0110] Synthesis of 8-methyl-4-amino-7-(2-bromoethoxy)coumarin

[0111]

[0112] 47.73 mg (0.25 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 69.7 mg (0.5 mmol) of anhydrous potassium carbonate, and 40.7 mg (0.125 mmol) of TBAB were dissolved in acetone (3 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to reflux at 56 °C and stirred for 20 min. 159.6 mg (0.75 mmol) of 1,2-dibromoethane was then added. The reaction was stopped after 26 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3a.

[0113] 1H NMR (400MHz, CD3OD_SPE) δ7.74(s,1H),7.01(s,1H),5.30(s,1H),4.46(s,2H),3.79(s,2H),2.32(s,3H).

[0114] Example 3

[0115] Synthesis of 8-methyl-4-amino-7-(3-bromopropoxy)coumarin

[0116]

[0117] 95.5 mg (0.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 139.5 mg (1 mmol) of anhydrous potassium carbonate, and 86.2 mg (0.25 mmol) of TBAB were dissolved in acetone (4 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to reflux at 56 °C and stirred for 20 min. 201.3 mg (1 mmol) of 1,3-dibromopropane was then added. The reaction was stopped after 46 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3b.

[0118] 1 H NMR(400MHz,CD3OD_SPE)δ7.74(d,J=8.9Hz,1H),7.01(d,J=8.9Hz,1H),5.29(s,1H), 4.18(t,J=5.9Hz,2H),3.58(t,J=6.4Hz,2H),2.29(s,3H),2.07(dd,J=8,6.5Hz,2H).

[0119] Example 4

[0120] Synthesis of 8-methyl-4-amino-7-(4-bromobutoxy)coumarin

[0121]

[0122] 47.7 mg (0.25 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 69.8 mg (0.5 mmol) of anhydrous potassium carbonate, and 39.9 mg (0.125 mmol) of TBAB were dissolved in acetone (3 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to 56 °C and refluxed. After stirring for 20 min, 107.7 mg (0.5 mmol) of 1,4-dibromobutane was added. The reaction was stopped after 21 h. The target product was separated, and the solid was washed with ethyl acetate. Ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3c.

[0123] 1 H NMR (400MHz, CD3OD_SPE) δ7.74(d,J=8.9Hz,1H),7.01(d,J=8.9Hz,1H),5.29(s,1H),4.18( t,J=5.9Hz,2H),3.58(t,J=6.4Hz,2H),2.29(s,3H),2.11(dd,J=8,6.9Hz,2H),2.03(m,2H).

[0124] Example 5

[0125] Synthesis of 8-methyl-4-amino-7-(3-methylbut-2-enoxy)coumarin

[0126]

[0127] 286.9 mg (1.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 442.1 mg (3 mmol) of anhydrous potassium carbonate, and 256.7 mg (0.8 mmol) of TBAB were dissolved in acetone (5 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to 56 °C and refluxed. After stirring for 20 min, 1.616 g (7.5 mmol) of 1,4-dibromo-2-butene was added. The reaction was stopped after 38 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and the target product was obtained by column chromatography.

[0128] 1 H NMR (400MHz, CD3OD_SPE) δ8.53 (s, 1H), 8.09 (d, J = 9.1Hz, 1H), 7.53 (s, 1H), 7.03 (s,1H),5.52(s,1H),3.96–3.75(m,2H),3.55(dd,J=12.7,6Hz,2H),1.66(s,3H).

[0129] Example 6

[0130] Synthesis of 8-methyl-4-amino-7-(2-bromohexanoyloxy)coumarin

[0131]

[0132] 157.4 mg (0.8 mmol) of 2-bromohexanoic acid and 0.55 mL (8 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C, followed by absorption of SO2 gas using a tail gas absorber. The mixture gradually turned into a pale yellow liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-bromohexanoyl chloride was dissolved in 2.5 mL of acetone with 95.4 mg (0.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C. The reaction was stopped after 32 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3e was obtained by column chromatography.

[0133] 1 H NMR(400MHz,CD3OD_SPE)δ7.07(d,J=9.0Hz,1H),6.73–6.66(m,1H),5.51(s,1H),4.2 2(s,1H),2.27(s,3H),1.96(t,J=4.7Hz,2H),1.35(s,2H),1.26(s,3H),1.11(s,2H). 13 C NMR (101MHz, CD3OD_SPE) δ164.87,164.00,162.24,154.67,153.92,125.06,123.40,120.52,116.11,87.81,46.79,31.47,26.34,23.19,14.25.

[0134] Example 7

[0135] Synthesis of 8-methyl-4-amino-7-(2-methylpentanoyloxy)methylcoumarin

[0136]

[0137] 1.395 g (12 mmol) of 2-methylpentanoic acid and 0.73 mL (12 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a dark brown liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-methylpentanoyl chloride was dissolved in acetone (6 mL) with 289.1 mg (1.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C and then the reaction was stopped after 35 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3f was obtained by column chromatography.

[0138] 1 H NMR (400MHz, CD3OD_SPE) δ7.68(dd,J=4,3.4Hz,1H),6.89(d,J=9.2Hz,1H),5.49(s,1H),2.66(s,1 H), 1.91 (s, 3H), 1.55 (d, J = 3.5Hz, 2H), 1.36 (d, J = 4.9Hz, 2H), 1.23 (s, 3H), 0.92 (d, J = 7.2Hz, 3H). 13 C NMR (101MHz, CD3OD_SPE) δ171.38,165.30,161.37,152.65,150.85,130.49,129.87,124.52,112.66,88.82,40.18,30.74,24.03,15.15,14.39.

[0139] Example 8

[0140] Synthesis of 8-methyl-4-amino-7-(2-methylhexanoyloxy)coumarin

[0141]

[0142] 1.573 g (12 mmol) of 2-methylhexanoic acid and 2.48 mL (20 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a dark brown liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-methylpentanoyl chloride was dissolved in acetone (5 mL) with 313.3 mg (1.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C and then the reaction was stopped after 40 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product (3 g) was obtained by column chromatography.

[0143] 1 H NMR (400MHz, CD3OD_SPE) δ8.16(d,J=8.8Hz,1H),7.10(d,J=8.8Hz,1H),5.55(s,1H),3.88(s,1 H),2.70(s,2H),2.30(s,3H),1.34(d,J=7.2Hz,2H),1.24(d,J=6.9Hz,2H),0.94–0.87(m,3H). 13 C NMR(101MHz,CD3OD_SPE)δ170.92,164.74,162.17,154.60,153.97,125.07 ,123.53,120.52,116.42,87.71,38.51,31.48,30.74,26.34,15.16,14.43.

[0144] Example 9

[0145] Synthesis of 8-methyl-4-amino-7-(piperidineethoxy)coumarin

[0146]

[0147] 48 mg (0.25 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 92.6 mg (0.5 mmol) of piperidine hydrochloride, 42.2 mg (0.75 mmol) of potassium hydroxide, 31.9 mg (0.1 mmol) of TBAB, and 41.1 mg (0.25 mmol) of potassium iodide were dissolved in acetone (3 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to reflux at 56 °C. The reaction was stopped after 31 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product was obtained by column chromatography for 3 h.

[0148] 1 H NMR (400MHz, CD3OD_SPE) δ7.75(d,J=8.9Hz,1H),7.02(d,J=9.0Hz,1H),5.29(s,1H),4.30(t,J=5.5Hz,2H ), 2.29 (s, 3H), 1.68 (s, 2H), 1.53 (d, J = 5.1Hz, 1H), 1.43 (dd, J = 7.36, 7.36Hz, 2H), 1.28 (d, J = 14.6Hz, 4H).

[0149] Example 10

[0150] Synthesis of 8-methyl-4-amino-7-(2-piperidinoxy)coumarin

[0151]

[0152] 1.006 g (8 mmol) of piperidinecarboxylic acid and 1.7 mL (8 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a dark brown liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained piperidinecarboxylic acid chloride was dissolved in acetone (7 mL) with 381.6 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C and then the reaction was stopped after 29 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected and evaporated to dryness by rotary evaporation. The target product 3i was obtained by column chromatography.

[0153] 1 H NMR (400MHz, CD3OD_SPE) δ8.06(d,J=9.0Hz,1H),7.01(d,J=9.2Hz,1H),5.52(s,1H),2.68(s,1H),2.27(s,3H),1.58(s,4H),1.56(s,4H). 13 C NMR (101MHz, CD3OD_SPE) δ184.35,171.63,169.29,156.58,155.89,124.85,123.25,117.83,87.29,45.11,38.58,26.34.

[0154] Example 11

[0155] Synthesis of 8-methyl-4-amino-7-(3-furanoyloxy)coumarin

[0156]

[0157] 168.2 mg (1.5 mmol) of 3-furoic acid and 1.5 mL (12 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C, followed by absorption of SO2 gas using a tail gas absorber. The mixture gradually turned into a dark brown liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained 3-furoyl chloride was dissolved in 2.5 mL of acetone with 382.1 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C, and after 8 hours, 1 mL of triethylamine was added. The reaction was stopped after 24 hours. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3j.

[0158] 1 H NMR (400MHz, CD3OD_SPE) δ8.09(s,1H),7.81(d,J=8.8Hz,1H),7.55(s,1H),6.92(s,1H),6.72(s,1H),5.39(s,1H),2.27(s,3H). 13 C NMR (101MHz, CD3OD_SPE) δ166.44,162.13,154.27,153.15,150.92,149.33,146.22,145.37,121.62,120.69,120.39,119.28,110.79,84.36.

[0159] Example 12

[0160] Synthesis of 8-methyl-4-amino-7-(malonoformyloxy)coumarin

[0161]

[0162] 708.5 mg (6 mmol) of potassium monomethyl malonate and 0.4 mL (6 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a pale yellow liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained formyl malonate chloride (i.e., methyl malonate chloride) was dissolved in acetone (4 mL) with 383.2 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C and then the reaction was stopped after 25 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3K was obtained by column chromatography.

[0163] 1H NMR (400MHz, CD3OD_SPE) δ7.64 (dd, J=9.24, 9.1Hz, 1H), 6.79–6.58 (m, 1H), 5.3 8(s,1H),3.78(d,J=9.3Hz,3H),3.48(q,J=7.0Hz,2H),2.12(d,J=19.2Hz,3H). 13 CNMR(101MHz,CD3OD_SPE)δ170.41,168.67,165.96,161.17,154.17,150.37,122.79,118.09,86.10,56.84,31.53.

[0164] Example 13

[0165] Synthesis of 8-methyl-4-amino-7-(3-hydroxyhexanoyloxy)coumarin

[0166]

[0167] 392.4 mg (3 mmol) of methyl-3-hydroxyhexanoic acid and 1.1 mL (15 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a dark brown liquid. After reacting for 5 hours, thionyl chloride was removed by vacuum distillation. The obtained malonyl chloride (i.e., methyl malonate chloride) was dissolved in acetone (4 mL) with 287.5 mg (1.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1. The mixture was heated to reflux at 56 °C and then the reaction was stopped after 36 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3 L was obtained by column chromatography.

[0168] 1 H NMR(600MHz,MeOD)δ7.56(d,J=8.8Hz,1H),6.74(d,J=8.8Hz,1H),5.34(s,1H),3.65(s ,1H),2.22(s,3H),2.18–2.13(m,2H),1.39(s,2H),1.36(d,J=6.7Hz,2H),1.21(s,3H).

[0169] Example 14

[0170] Synthesis of 8-methyl-4-amino-7-(3-methyl-2-enoxy)coumarin

[0171]

[0172] 47.75 mg (0.25 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 69.8 mg (0.5 mmol) of anhydrous potassium carbonate, and 41.3 mg (0.125 mmol) of TBAB were dissolved in acetone (2 mL) and then added to a 50 mL round-bottom flask. The mixture was heated to 56 °C and refluxed. After stirring for 20 min, 226.7 mg (1.5 mmol) of 3,3-dimethylallyl bromide was added. After 21 h, 74.9 mg (0.5 mmol) of 3,3-dimethylallyl bromide was added. The reaction was stopped after 31 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3m.

[0173] 1 H NMR (400MHz, CD3OD_SPE) δ7.64(d,J=8.8Hz,1H),6.80(d,J=8.8Hz,1H),5.11(s,1H),4.31(s,1H),2.26(s,3H),1.80(d,J=8.2Hz,6H). 13 C NMR (101MHz, CD3OD_SPE) δ165.90,159.74,151.29,147.39,132.35,129.87,120.91,120.54,114.31,112.40,89.12,66.66,24.95,20.26.

[0174] Example 15

[0175] Synthesis of 8-methyl-4-amino-7-(2-bromopentanoyloxy)coumarin

[0176]

[0177] 1.0883 g (6 mmol) of 2-bromopentanoic acid and 1.3 mL (18 mmol) of thionyl chloride were added to a 50 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a pale yellow liquid. After reacting for 6 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-bromopentanoyl chloride and 382 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1 were dissolved in acetone (7 mL) and heated to reflux at 56 °C. The reaction was stopped after 12 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3n was obtained by column chromatography.

[0178] 1H NMR(600MHz,MeOD)δ8.08(d,J=9.0Hz,1H),7.09(d,J=9.0Hz,1H),5.53(s,1H),2 .30(s,3H),2.21(d,J=4.9Hz,2H),1.42(dd,J=12.48,12.6Hz,3H),1.28(s,2H). 13 CNMR(151MHz,MeOD)δ163.99,162.26,154.73,154.68,153.93,125.05,123.35,120.55,116.08,87.83,38.47,31.47,26.34,8.19,3.24.

[0179] Example 16

[0180] Synthesis of 8-methyl-4-amino-7-(2-methylbutyryloxy)coumarin

[0181]

[0182] 2.0562 g (20 mmol) of 2-methylbutyric acid and 5.02 mL (50 mmol) of thionyl chloride were added to a 100 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a pale yellow liquid. After reacting for 6 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-methylbutyryl chloride and 957.2 mg (5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1 were dissolved in acetone (15 mL) and heated to reflux at 56 °C. The reaction was stopped after 18 hours. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 30 was obtained by column chromatography.

[0183] 1 H NMR (600MHz, MeOD) δ8.07(d,J=9.0Hz,1H),7.08(d,J=9.0Hz,1H),5.53(s,1H),2.68(s,2H),2.29(s,3H),2.23(s,1H),1.57(s,3H),1.55(s,3H). 13 C NMR (151MHz, MeOD) δ162.91,160.85,153.49,153.26,152.57,123.62,121.89,119.17,114.83,86.40,37.09,30.06,24.93,6.90,6.78.

[0184] Example 17

[0185] Synthesis of 8-methyl-4-amino-7-(2-bromobutyryloxy)coumarin

[0186]

[0187] 1.0129 g (6 mmol) of 2-bromobutyric acid and 1.3 mL (18 mmol) of thionyl chloride were added to a 100 mL round-bottom flask. The mixture was heated to reflux at 70 °C and then connected to a tail gas absorption device to absorb SO2 gas. The mixture gradually turned into a pale yellow liquid. After reacting for 6 hours, thionyl chloride was removed by vacuum distillation. The obtained 2-bromobutyryl chloride and 386.7 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1 were dissolved in acetone (10 mL) and heated to reflux at 56 °C. The reaction was stopped after 5 hours. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, and the organic phase was evaporated to dryness by rotary evaporation. The target product 3p was obtained by column chromatography.

[0188] 1 H NMR (600MHz, MeOD) δ8.07(d,J=9.0Hz,1H),7.08(d,J=9.0Hz,1H),5.53(s,1H),4.21(s,1H),2.29(s,3H),2.22(d,J=12.3Hz,2H),1.46–1.41(m,3H). 13 C NMR (151MHz, MeOD) δ164.13,162.89,162.24,154.67,153.94,125.03,123.32,120.56,116.14,87.81,57.91,31.47,26.34,8.19.

[0189] Example 18

[0190] Synthesis of 8-methyl-4-amino-7-allyloxycoumarin

[0191]

[0192] 382.6 mg (2 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1, 314.6 mg (2.25 mmol) of anhydrous potassium carbonate, and 324.9 mg (1 mmol) of TBAB were dissolved in 10 mL of acetone and then added to a 50 mL round-bottom flask. The mixture was heated to 56 °C and refluxed under condensation. After stirring for 20 min, 488.4 mg (4 mmol) of 3-bromopropene was added. The reaction was stopped after 7 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3q.

[0193] 1 H NMR(600MHz,MeOD)δ7.71(d,J=8.9Hz,1H),6.98(d,J=9.0Hz,1H),6.11(dd,J=11.9,5.4Hz, 1H),5.45(d,J=18.7Hz,1H),5.30(d,J=11.7Hz,1H),5.27(s,1H),4.64(s,2H),2.28(s,3H). 13 C NMR (151MHz, MeOD) δ167.40,161.12,159.44,154.18,134.42,121.83,117.72,115.05,109.18,109.09,82.91,70.37,8.46.

[0194] Example 19

[0195] Synthesis of 8-methyl-4-amino-7-benzyloxycoumarin

[0196]

[0197] 287.2 mg (1.5 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1 and 552 mg (4 mmol) of anhydrous potassium carbonate were dissolved in DMF (9 mL) and then added to a 50 mL round-bottom flask. The reaction system was evacuated to a vacuum and protected with N2. The mixture was heated to 60 °C and refluxed, stirred for 20 min, and then 0.28 mL (2.25 mmol) of benzyl bromide was added. The reaction was stopped after 4 h. The solid was washed with ethyl acetate, and ethyl acetate was extracted three times with saturated sodium chloride solution. The organic phase was collected, evaporated to dryness by rotary evaporation, and separated by column chromatography to obtain the target product 3r.

[0198] 1H NMR (600MHz, DMSO) δ7.73 (d, J = 8.9 Hz, 1H), 7.33 (ddd, J = 7.38, 7.74, 7.32 Hz, 5H), 7.02 (d, J = 9.0 Hz, 1H), 5.18 (s, 2H), 5.01 (s, 1H), 2.12 (s, 3H). 13 C NMR (151MHz, DMSO) δ171.47,168.35,165.60,162.22,146.38,138.05,137.43,136.94,130.58,122.22,117.45,117.29,91.38,79.29,17.89.

[0199] Example 20

[0200] Synthesis of 8-hydroxy-2,4,7-trimethylcoumarin[3,4-b]pyridine

[0201]

[0202] 51.9 mg (0.5 mmol) of 2,4-pentanedione was stirred with 0.13 mL (2.5 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 47.4 mg (0.25 mmol) of 7-hydroxy-4-amino-8-methylcoumarin prepared in Example 1 was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 3 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4.

[0203] 1 H NMR (400MHz, CD3OD_SPE) δ8.20(s,1H),7.16(s,1H),6.80(s,1H),2.75(s,3H),2.61(s,3H),2.27(s,3H). 13 C NMR (101MHz, CD3OD_SPE) δ171.58,164.56,161.47,159.02,153.24,152.94,124.86,122.75,121.89,111.55,111.35,110.91,21.38,13.05.

[0204] Example 21

[0205] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-methylbutyrate)

[0206]

[0207] 439 mg (4.36 mmol) of 2,4-pentanedione was mixed with 0.7 mL (10.9 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 300 mg (1.09 mmol) of 4-amino-2-oxo-7-(2-methylbutyrate)-8-methylcoumarin was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 20 h. The mixture was quenched with 10 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4a.

[0208] 1 H NMR(600MHz,MeOD)δ8.08(d,J=9.0Hz,1H),7.09(d,J=9.0Hz,1H),5.54(t,J=1.4H z,1H),2.69(d,J=1.3Hz,2H),2.30(s,3H),2.24(s,1H),1.58(s,3H),1.56(s,3H). 13 C NMR(151MHz,MeOD)δ171.34,165.31,159.60,154.06,153.38,151.64,129.25 ,123.53,123.34,120.98,120.70,112.49,31.17,26.38,20.52,14.46,8.23.

[0209] Example 22

[0210] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-bromobutyrate)

[0211]

[0212] 366 mg (3.6 mmol) of 2,4-pentanedione was stirred with 0.5 mL (9.2 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 312 mg (0.92 mmol) of 4-amino-2-oxo-7-(2-bromobutyrate)-8-methylcoumarin was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 20 h. The mixture was quenched with 10 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4b.

[0213] The nuclear magnetic resonance (NMR) spectroscopy data are as follows:

[0214] 1H NMR (600MHz, MeOD) δ7.67(d,J=8.9Hz,1H),6.87(d,J=8.7Hz,1H),6.76(d,J=8.8Hz,1H),4.59 (s,1H),2.28(s,3H),2.22(s,3H),2.15(d,J=1.4Hz,3H),1.94(s,2H),0.89(d,J=7.0Hz,3H). 13 C NMR (151MHz, MeOD) δ168.98,164.13,162.24,154.67,153.94,125.03,123.32,120.56,116.14,87.81,50.72,26.34,8.19.

[0215] Example 23

[0216] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-bromohexanoate)

[0217]

[0218] 441.1 mg (4.35 mmol) of 2,4-pentanedione was stirred with 0.46 mL (8.7 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 320.7 mg (0.87 mmol) of 4-amino-2-oxo-7-(2-bromohexanoate)-8-methylcoumarin (compound 3e) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 12 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4e.

[0219] 1 H NMR (600MHz, MeOD) δ7.57(d,J=8.8Hz,1H),6.89(d,J=8.6Hz,1H),6.75(d,J=8.8Hz,1H),4.29(t,J=6.6Hz,1H),2 .54(d,J=6.8Hz,3H),2.27(s,3H),2.13(s,3H),2.25-2.20(m,2H),1.39(s,2H),1.24(s,2H),0.91-0.88(m,3H).

[0220] Example 24

[0221] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-methylvalerate)

[0222]

[0223] 524 mg (5.15 mmol) of 2,4-pentanedione was mixed with 0.55 mL (10.3 mmol) of 70% concentrated sulfuric acid and stirred at 120 °C for 10 min. Then, 298 mg (1.03 mmol) of 4-amino-2-oxo-7-(2-methylvalerate)-8-methylcoumarin (compound 3f) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 10 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until solid precipitated again. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4f.

[0224] 1 H NMR (600MHz, MeOD) δ7.57(d,J=8.8Hz,1H),6.86(d,J=8.6Hz,1H),6.75(d,J=8.8Hz,1H),2.54(s,1H),2.49(d,J=12. 1Hz, 3H), 2.23 (s, 3H), 2.15 (d, J = 6.4Hz, 3H), 1.39 (s, 2H), 1.36 (d, J = 6.7Hz, 2H), 1.28 (s, 3H), 0.96 (t, J = 7.0Hz, 3H). 13 C NMR(151MHz,MeOD)δ178.17,173.84,170.86,156.31,145.25,144.18,132.28,131.87,1 30.84,125.03,121.69,120.69,30.75,29.93,24.22,20.55,17.29,15.18,14.45,8.33.

[0225] Example 25

[0226] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-methylhexanoate)

[0227]

[0228] 400.43 mg (4 mmol) of 2,4-pentanedione was stirred with 0.52 mL (9.7 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 309.6 mg (0.97 mmol) of 4-amino-2-oxo-7-(2-methylhexanoate)-8-methylcoumarin (3 g) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 6 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain 4 g of the target product.

[0229] 1 H NMR (600MHz, MeOD) δ7.57(d,J=8.8Hz,1H),6.87(d,J=8.7Hz,1H),6.75(d,J=8.8Hz,1H),2.67(d,J=18.8Hz,1H),2.23(s,3H),2. 15(s,3H),1.98–1.71(m,3H),1.53(dd,J=13.1,9.4Hz,2H),1.34(s,2H),1.28(s,2H),1.21(t,J=7.0Hz,3H),0.95–0.83(m,3H). 13 C NMR(151MHz,MeOD)δ166.32,165.37,160.04,153.38,151.71,151.23,130.85,127.15,1 26.87,123.00,120.72,112.44,30.75,29.92,21.38,20.53,15.21,14.43,14.41,8.29.

[0230] Example 26

[0231] Synthesis of 2,4,7-trimethyl-8-(2-piperidineethoxy)coumarin[3,4-b]pyridine

[0232]

[0233] 110.1 mg (1.1 mmol) of 2,4-pentanedione was stirred with 0.23 mL (4.4 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 132 mg (0.44 mmol) of 4-amino-7-(piperidin-N-)ethoxy-8-methylcoumarin (3 h) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The reaction was quenched with 6 mL of ice-cold distilled water, and a solid precipitated. The target product was separated to obtain 4 h.

[0234] 1H NMR (400MHz, CD3OD_SPE) δ8.39 (d, J=8.9Hz, 1H), 7.22 (s, 1H), 7.09–7.02 (m, 1H), 2.90 (t, J= 5.5Hz,3H),2.76(s,2H),2.64(s,3H),2.50(s,3H),2.31(d,J=8.7Hz,4H),1.71–1.62(m,6H).

[0235] Example 27

[0236] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-piperidinecarboxylate

[0237]

[0238] 1.325 g (13.2 mmol) of 2,4-pentanedione was stirred with 1.8 mL (33.4 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 1.0094 g (3.34 mmol) of 4-amino-2-oxo-7-(piperidinecarboxylate)-8-methylcoumarin (3i) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 15 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4i.

[0239] 1 H NMR(600MHz,MeOD)δ7.55(d,J=8.8Hz,1H),6.83(s,1H),6.74(d,J=8.8Hz,1H),2.79(d,J=3.2 Hz,4H),2.47(t,J=4.5Hz,3H),2.39(s,1H),2.27(d,J=5.2Hz,3H),2.21(s,3H),1.45(s,4H). 13 C NMR(151MHz,MeOD)δ164.23,159.52,155.13,155.01,153.40,152.33,120.80,1 19.47,119.07,112.15,108.04,98.22,44.94,32.39,28.79,28.22,21.49,8.22.

[0240] Example 28

[0241] Synthesis of methyl 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-malonate

[0242]

[0243] 1.2050 g (12 mmol) of 2,4-pentanedione was stirred with 1.52 mL (28.6 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 831.9 mg (2.86 mmol) of 4-amino-2-oxo-7-(methyl malonate)-8-methylcoumarin (3k) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 4 h. The mixture was quenched with 12 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4j.

[0244] 1 H NMR(600MHz,MeOD)δ7.62(s,1H),7.06(d,J=8.8Hz,1H),6.93(d,J=8.8Hz,1H),3. 74(s,1H),3.08(s,1H),2.26(t,J=5.8Hz,3H),2.21(d,J=3.2Hz,3H),2.14(s,3H). 13 C NMR(151MHz,MeOD)δ170.36,165.31,162.15,159.60,153.38,151.63,150.63,12 5.22,124.38,123.34,120.70,112.46,112.24,51.06,29.92,26.39,20.52,8.25.

[0245] Example 29

[0246] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(3-hydroxyhexanoate)

[0247]

[0248] 779.5 mg (7.7 mmol) of 2,4-pentanedione was stirred with 1.03 mL (19.2 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 608 mg (1.92 mmol) of 4-amino-2-oxo-7-(3-hydroxyhexanoate)-8-methylcoumarin (3L) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 8 h. The mixture was quenched with 12 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4K.

[0249] 1H NMR (600MHz, MeOD) δ8.54(s,1H),7.56(d,J=8.8Hz,1H),6.74(d,J=8.8Hz,1H),3.65(s ,1H),2.22(s,3H),1.98–1.79(m,8H),1.39(s,2H),1.28(s,2H),0.90(t,J=6.9Hz,3H).

[0250] Example 30

[0251] Synthesis of 2,4,7-trimethyl-8-(3-methyl-2-enoxy)coumarin[3,4-b]pyridine

[0252]

[0253] 4.5127 g (45 mmol) of 2,4-pentanedione was stirred with 5.3 mL (100 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 2.8002 g (10 mmol) of 4-amino-7-(3-methylbutyl-2-enoxy)-8-methylcoumarin (3 mL) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 20 h. The mixture was quenched with 12 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain 4 μL of the target product.

[0254] 1 H NMR (600MHz, MeOD) δ7.60(d,J=8.8Hz,1H),6.77(d,J=8.8Hz,1H),2.54(t,J=6.7Hz,3H),2.24–2.23(m,3H),1.72(t,J=6.7Hz,3H),1.32–1.32(m,6H). 13 C NMR(151MHz,MeOD)δ166.10,161.95,159.68,157.82,153.35,151.51,135.18,125.13,12 3.49,122.65,120.42,112.70,112.34,108.02,88.67,34.58,28.53,27.70,19.35,8.29.

[0255] Example 31

[0256] Synthesis of 2,4,7-trimethylcoumarin[3,4-b]pyridine-8-(2-bromopentanoate)

[0257]

[0258] 598.8 mg (5.95 mmol) of 2,4-pentanedione was stirred with 0.63 mL (11.9 mmol) of 70% concentrated sulfuric acid at 120 °C for 10 min. Then, 420.1 mg (1.19 mmol) of 4-amino-2-oxo-7-(2-bromopentanoate)-8-methylcoumarin (3n) was added, and the mixture was heated to 125 °C and refluxed. The reaction was stopped after 20 h. The mixture was quenched with 10 mL of ice-cold distilled water, and a solid precipitated. The solid was collected by vacuum filtration, and the pH of the filtrate was adjusted with 30% and 10% NaOH solutions until a solid precipitated. The solid was collected by vacuum filtration and separated by column chromatography to obtain the target product 4m.

[0259] 1 H NMR (600MHz, MeOD) δ7.67(d,J=8.9Hz,1H),7.07(s,1H),6.75(s,1H),4.29(t,J=6.6Hz,1H),2.28( t,J=5.6Hz,3H),2.23(d,J=2.6Hz,3H),2.21(s,3H),2.15(s,2H),1.37(s,2H),0.91–0.88(m,3H). 13 C NMR (151MHz, MeOD) δ165.65,163.99,162.26,154.68,153.93,125.05,123.35,120.55,116.08,87.83,38.47,31.47,26.34,13.04,8.19.

[0260] The antibacterial tests were conducted on a series of coumarin compounds synthesized in the above embodiments, as detailed below:

[0261] 1. Materials

[0262] 1.1 Experimental strains

[0263] As shown in Table 1, four standard bacterial strains were used in this part of the experiment. 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.

[0264] Table 1

[0265]

[0266]

[0267] 1.2 Medicines and Reagents

[0268] Positive controls: carbendazim purchased from CATO; chlorothalonil purchased from Beijing Bailingwei Technology Co., Ltd.; dimethyl sulfoxide (DMSO) purchased from Tianjin Yongsheng Fine Chemical Co., Ltd.

[0269] 1.3 Main Liquid Formulation

[0270] 1.3.1 PDA culture medium

[0271] Add 200g of peeled potatoes to 1000mL of distilled water and cook until soft but not mushy. Collect the filtrate. Add 18g of agar and 20g of glucose to the filtrate, stir well, and bring the volume to 1000mL with distilled water. Autoclave.

[0272] 1.3.2 Preparation of Compound Solutions

[0273] Accurately weigh 24 mg of the synthesized coumarin series compounds, add 1200 μL of DMSO, sonicate to dissolve, and sterilize under a UV sterilizer for 15 min to obtain a 200 μg / mL concentration solution.

[0274] 2 methods

[0275] 2.1 Determination of initial screening inhibitory concentration

[0276] The inhibitory effects of the synthesized coumarin series compounds, carbendazim, and chlorothalonil on four fungi were determined using the mycelial growth inhibition method. PDA medium was poured into sterile Petri dishes, 10 mL per dish. A 200 μg / mL solution was then added to the medium, and the mixture was thoroughly mixed to prepare the drug-containing medium. Using a 7 mm diameter punch, activated mycelial cakes of different test strains were collected and inoculated into the center of the drug-containing medium plates. Each compound was tested in triplicate, with three media containing only carbendazim or chlorothalonil serving as positive controls. The plates were incubated at 25°C for 4 days. Colony diameters were measured using the cross-hatching method, and the relative inhibition rate at a concentration of 200 μg / mL was calculated. The results at 48, 72, and 96 h are shown in Tables 2, 3, and 4.

[0277] Table 2

[0278]

[0279]

[0280] Table 3

[0281]

[0282] Table 4

[0283]

[0284] As can be seen from the results in Tables 2-4, most compounds showed the best antibacterial effect after 48 hours. Twelve compounds showed antibacterial activity of more than 50% against Alternaria alternata and Alternaria solanacea, indicating good antibacterial effect. In particular, compound 3a showed antibacterial activity of more than 50% against all four plant pathogens, indicating that the coumarin compounds of the present invention have broad-spectrum antibacterial activity.

[0285] Under the treatment of these four plant pathogenic fungi with existing fungicides such as carbendazim or chlorothalonil, which have been proven to have antibacterial effects, these fungi will inevitably show inhibitory effects. Using these fungi as a positive control group, the coumarin compounds of the present invention, under the same treatment conditions, also have a complete or partial inhibitory effect on the above four plant pathogenic fungi, indicating that the coumarin compounds of the present invention also have good antibacterial effects. These compounds can be used directly as antibacterial agents or as lead compounds for new skeleton fungicides. There are few reports on the antibacterial effect of C-4 amino substitution of coumarin.

[0286] It should be noted that carbendazim or chlorothalonil have a large structural difference from the coumarin compounds of this invention, and are not used as objects for comparison with the antibacterial rate of the compounds synthesized in this patent. Only carbendazim and chlorothalonil are used as positive controls, which can fully demonstrate the reliability of this experiment.

[0287] 2.2EC 50 Measurement

[0288] EC 50 The value refers to the half-maximal inhibitory concentration (MCC). The inhibitory effect of compounds with good initial antibacterial effects on four fungi was determined using the mycelial growth inhibition method. PDA medium was poured into sterile petri dishes, 10 mL per dish. Based on the degree of inhibition, solutions of different concentrations were added to the medium and mixed to prepare drug-containing media with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.125 μg / mL, respectively. Using a 7 mm diameter punch, activated mycelial pellets of different test strains were collected and inoculated into the center of the drug-containing media plates. Each compound was tested in triplicate: three media containing only DMSO as controls (CK) and three media containing only carbendazim or chlorothalonil as positive controls. The plates were incubated at 25°C for 4 days. Colony diameters were measured using the cross-sectional method, and the EC was calculated based on the relative inhibition rate. 50 Value, EC 50 The measurement results are shown in Table 5.

[0289] Table 5 EC5 of compounds against Alternaria. 50 value

[0290]

[0291] Table 6. EC50 of compounds against Alternaria solanacearum50 value

[0292]

[0293]

[0294] Table 7. EC50 of compounds against Staphylococcus aureus 50 value

[0295]

[0296] Table 8. EC50 of compounds against Fusarium oxysporum 50 value

[0297]

[0298] Note: A 95% confidence interval means that there is a 95% probability that the measured EC50 value will fall within the interval of the measurement result.

[0299] Based on the above results, it can be seen that:

[0300] (1) In this invention, a higher relative inhibition rate indicates a better antibacterial effect. Among the coumarin compounds of this invention, 12 compounds exhibited strong bactericidal activity against Alternaria solanacea and Alternaria solani. Overall, the antibacterial activity at 48 h was generally higher than that at 72 h and 96 h. In addition, compound 4i achieved an inhibition rate of 86.43% against Alternaria solani at 48 h, and compound 4a achieved an inhibition rate of 69.76% against Alternaria solanacea at 72 h, and can be used as antibacterial agents.

[0301] (2) Compounds 3a and 4a of the present invention have strong antibacterial effects against Botrytis cinerea, Alternaria solanacea, Fusarium oxysporum and Alternaria, and exhibit good broad-spectrum antibacterial activity.

[0302] (3) In this invention, EC 50 The lower the value, the lower the toxicity. Experimental results show that the compound with the best activity against Alternaria is 4g (EC). 50 =30.92 μg / mL), compound 4a (EC 50 =39.84μg / mL), 4i (EC 50 =34.17μg / mL), 4f(EC) 50 The half-maximal inhibitory concentrations (IC50) of all compounds against Alternaria solanaceae (38.33 μg / mL) were below 40 μg / mL; the compound with the best activity against Alternaria solanaceae was 4m (EC50). 50 =14.41 μg / mL), compound 4b (EC 50 =21.86 μg / mL), compound 4l (EC) 50=27.93 μg / mL), compound 4f (EC 50 The half-maximal effective concentration (MCP) of 3a against Botrytis cinerea (29.76 μg / mL) was also below 30 μg / mL. 50 The value was 105.8 μg / mL, which is the EC50 value for Fusarium oxysporum. 50 The value was 105.2 μg / mL, and most compounds had EC values ​​of 105.2 μg / mL. 50 The value of R 2 The values ​​were all greater than 0.9, indicating a good linear correlation between the concentration of these compounds and their antibacterial effect.

[0303] Comparative Example 1

[0304]

[0305] The compound shown in Comparative Example 1 was tested under the same test conditions as the compounds in the examples of this invention, and the relative inhibition rate results after 96 hours are as follows:

[0306]

[0307] Compared with the compound shown in Comparative Example 1, compound 4a of the present invention has an additional methyl group at the C-4 position. The results of the relative inhibition rates of the two compounds show that the addition of an additional methyl group at the C-4 position can make the antibacterial effect of coumarin compounds better.

[0308] 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.

Claims

1. A coumarin compound selected from compounds represented by formula (II): The structure of R2 is as follows:

2. The method for preparing the coumarin compound according to claim 1, comprising the following steps: The preparation of the compound represented by formula (II) includes the following methods: Method 3: The starting materials, including the compound shown in formula (I) and a dialkyl ketone, are reacted in concentrated acid to produce the compound shown in formula (II); The compound represented by formula (I): R1 is selected from:

3. The method for preparing coumarin compounds according to claim 2, characterized in that: In method three, The dialkyl ketone is selected from 2,4-pentanedione; and / or, The concentrated acid is selected from concentrated sulfuric acid; and / or, The reaction temperature is 100-135℃; and / or, The reaction time is 3-6 hours; and / or, The molar ratio of the compound shown in formula (I) to the dialkyl ketone is 1:1-8; and / or, The ratio of the compound shown in formula (I) to concentrated acid is 1 mmol: 0.3-2 mL.

4. The method for preparing coumarin compounds according to claim 2, characterized in that: In method three, The concentrated sulfuric acid has a mass concentration of 70%-80%; and / or, The reaction temperature is 120-130℃; and / or, The reaction time is 4-5 hours; and / or, The molar ratio of the compound shown in formula (I) to the dialkyl ketone is 1:1-4.

5. The use of a coumarin compound of claim 1 in the treatment of plant fungi.

6. An antibacterial agent comprising the coumarin compound of claim 1.

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