Pyridone type coumarin derivative as well as preparation method and application thereof

Abstract

The invention discloses a pyridone type coumarin derivative as well as a preparation method and application thereof. The invention autonomously designs and synthesizes a novel pyridone type coumarin derivative. Experimental results show that the pyridone type coumarin derivative has a remarkable inhibition effect on KYSE-70 and KYSE-150 cells, and the pyridone type coumarin derivative has a very good application value in anti-inflammatory, anti-virus, antibacterial or anti-cancer aspects.

Description

Technical Field

[0001] The present invention relates to the technical field of coumarin derivatives. Further, it relates to pyridone-type coumarin derivatives, their preparation methods and applications. Background Art

[0002] Natural products and their complex molecular frameworks provide researchers with a series of unknown chemical types to discover new chemical probes and drugs. Therefore, natural products are considered as a source of inspiration for drug design, and many existing anti-cancer drugs have been discovered from natural products, such as paclitaxel, novobiocin, etc.

[0003] Coumarin is a naturally occurring heterocyclic compound with good broad-spectrum biological activities, including anti-inflammatory, anti-viral, antibacterial, and anti-cancer. Further modification of existing coumarins is to further improve their above-related properties. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention provides pyridone-type coumarin derivatives, their preparation methods and applications. We designed and synthesized new coumarin derivatives and preliminarily studied their inhibitory effects on KYSE-70 and KYSE-150 cells. We found that it has significant inhibitory effects and further explored its related sites through molecular virtual docking technology.

[0005] One object of the present invention is to provide a pyridone-type coumarin derivative, and the pyridone-type coumarin derivative is selected from the following general structural formula:

[0006]

[0007] Formula 1; wherein, R1 in Formula 1 is selected from hydrogen, a substituted or unsubstituted carbonyl group;

[0008]

[0009] Formula 2; wherein, R2 in Formula 2 is selected from hydrogen, a substituted or unsubstituted carbonyl group;

[0010]

[0011] Formula 3; wherein, R3 in Formula 3 is selected from hydrogen, a substituted or unsubstituted carbonyl group;

[0012]

[0013] Formula 4; wherein, R4 in Formula 4 is selected from a hydroxyl group, a substituted alkoxy group, a substituted ester group; R5 is selected from hydrogen, an alkyl group.

[0014] Among the pyridone-type coumarin derivatives of the present invention, preferably,

[0015] R1 in the formula 1 is selected from hydrogen, wherein, R 1a is selected from alkenyl, substituted or unsubstituted phenyl;

[0016] Preferably, R 1a is selected from C2-C8 alkenyl; phenyl substituted by at least one of halogen, C1-C4 alkyl; phenyl;

[0017] More preferably, R 1a is selected from C2-C6 linear alkenyl, phenyl substituted by two halogens in the meta position, phenyl substituted by one halogen and one C1-C2 alkyl in the meta position, phenyl;

[0018] Even more preferably, the formula 1 is selected from the following compounds:

[0019]

[0020] In the pyridone-type coumarin derivatives of the present invention, preferably,

[0021] R2 in the formula 2 is selected from hydrogen, wherein, R 2a is selected from alkenyl, substituted or unsubstituted phenyl;

[0022] Preferably,

[0023] R 2a is selected from C2-C8 alkenyl; phenyl substituted by at least one of halogen, C1-C4 alkyl, fluoroalkyl or hydroxyl; phenyl;

[0024] More preferably, R 2a is selected from C3-C8 alkenyl, phenyl substituted by two halogens in the meta position, phenyl substituted by one halogen and one C1-C2 alkyl, phenyl substituted by three halogens, phenyl substituted by one fluoroalkyl, phenyl substituted by one fluoroalkyl and one halogen in the ortho position, phenyl substituted by one fluoroalkyl and one hydroxyl in the meta position, phenyl;

[0025] Even more preferably, the formula 2 is selected from the following compounds:

[0026]

[0027] In the pyridone-type coumarin derivatives of the present invention, preferably,

[0028] R3 in the formula 3 is selected from hydrogen, wherein, R 3a is selected from substituted or unsubstituted phenyl; substituted or unsubstituted thiophenyl;

[0029] Preferably,

[0030] R 3a Selected from fluoroalkyl-substituted phenyl, phenyl, thiophenyl;

[0031] More preferably, R 3a Selected from a fluoroalkyl-substituted phenyl, phenyl, thiophenyl;

[0032] Even more preferably, Formula 3 is selected from the following compounds:

[0033]

[0034] In the pyridone-type coumarin derivative of the present invention, preferably,

[0035] R4 in Formula 4 is selected from hydrogen, wherein, R 4a1 Selected from pyridyl with substituents; R 4a2 Selected from C3-C6 cycloalkyl, halogen-substituted thiophenyl, C1-C4 alkyl-substituted oxazolyl;

[0036] R5 is selected from hydrogen, C1-C4 alkyl;

[0037] Preferably, R 4a1 Selected from pyridyl with an alkylcarbonyl substituent; R 4a2 Selected from C3-C4 cycloalkyl, a halogen-substituted thiophenyl, C1-C2 alkyl-substituted oxazolyl; and / or,

[0038] R5 is selected from hydrogen, C1-C2 alkyl;

[0039] Even more preferably, Formula 4 is selected from the following compounds:

[0040]

[0041]

[0042] The second object of the present invention is to provide a method for preparing a pyridone-type coumarin derivative as described in the first object of the present invention, comprising the following steps:

[0043] Method 1: Reacting Compound 1 with R 1a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 1a in -H, R 1a corresponds to and is the same as R 1a described in any one of claims 1-5;

[0044] wherein, the structural formula of Compound 1 is:

[0045] Method 2: React compound 2 with R 2a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 2a -H is the same as R in any one of claims 1-5 2a correspondingly; 2a

[0046] Among them, the structural formula of compound 2 is:

[0047] Method 3: React compound 3 with R 3a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 3a -H is the same as R in any one of claims 1-5 3a correspondingly; 3a

[0048] Among them, the structural formula of compound 3 is:

[0049] Method 4:

[0050] Step (1) Under a protective atmosphere, heat and react compound 4 and a cyclization catalyst, and perform post-treatment to obtain compound 4a;

[0051] Optional step (2): React compound 4a with an alkali solution to obtain the pyridone-type coumarin derivative;

[0052] Optional step (3): React compound 4a with R 4a1 -X, a nucleophilic reagent, and an acid-binding agent in a solvent to obtain the pyridone-type coumarin derivative; R 4a1 -X is the same as R in any one of claims 1-5 4a1 correspondingly; X is a halogen; 4a1

[0053] Optional step (4): React compound 4a with R 4a2 -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 4a2 -H is the same as R in any one of claims 1-5 4a2 correspondingly; 4a2

[0054] Among them, the structural formula of compound 4 is:

[0055] The structural formula of compound 4a is:

[0056] ​​​​In the preparation method of the pyridone-type coumarin derivative of the present invention, preferably,

[0057] In Method 1, Method 2, Method 3, and Method 4, the condensing agent in step (4) is selected from at least one of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or DCC (N,N-dicyclohexylcarbodiimide); and / or,

[0058] In Method 1, Method 2, Method 3, and Method 4, the basic catalyst in step (4) is selected from 4-dimethylaminopyridine (DMAP); and / or,

[0059] In Method 1, Method 2, Method 3, and Method 4, the solvent in step (4) is selected from at least one of dichloromethane, DMF, diethyl ether, or tetrahydrofuran; and / or,

[0060] In Method 4, the cyclization catalyst in step (1) is selected from at least one of polyphosphoric acid (PPA) or Eaton's reagent; and / or,

[0061] The base in the lye in step (2) is selected from inorganic bases, preferably selected from at least one of NaOH, sodium carbonate, or sodium bicarbonate; and / or,

[0062] The nucleophile in step (3) is selected from halide salts, preferably selected from at least one of KI or NaI; and / or,

[0063] The acid-binding agent in step (3) is selected from at least one of potassium carbonate, sodium carbonate, or sodium bicarbonate; and / or,

[0064] The solvent in step (3) is selected from at least one of DMF, dichloromethane, or diethyl ether.

[0065] In the preparation method of the pyridone-type coumarin derivative of the present invention, preferably,

[0066] In step (4) of Method 1, Method 2, Method 3, and Method 4,

[0067] The molar ratio of the condensing agent to the basic catalyst is independently (5-8):1; for example, 5:1, 6:1, 7:1, 8:1; and / or,

[0068] The mass-volume ratio of the condensing agent to the solvent is independently (2-20) mg:1 mL; for example, 2 mg:1 mL, 5 mg:1 mL, 10 mg:1 mL, 15 mg:1 mL, 20 mg:1 mL; and / or,

[0069] The molar ratio of Compound 1 to R 1a -H is 1:(2-4); for example, 1:2, 1:3, 1:4; and / or,

[0070] The molar ratio of Compound 1 to the condensing agent is 1:(2 - 6); for example, 1:2, 1:3, 1:4, 1:5, 1:6; and / or,

[0071] In Method 2, the molar ratio of Compound 2 to R 2a -H is 1:(2 - 4); for example, 1:2, 1:3, 1:4; and / or,

[0072] The molar ratio of Compound 2 to the condensing agent is 1:(2 - 6); for example, 1:2, 1:3, 1:4, 1:5, 1:6; and / or,

[0073] In Method 3, the molar ratio of Compound 3 to R 3a -H is 1:(2 - 4); for example, 1:2, 1:3, 1:4; and / or,

[0074] The molar ratio of Compound 3 to the condensing agent is 1:(2 - 6); for example, 1:2, 1:3, 1:4, 1:5, 1:6; and / or,

[0075] In step (4) of Method 4, the molar ratio of Compound 4a to R 4a2 -H is 1:(1 - 4); for example, 1:1, 1:2, 1:3, 1:4; and / or,

[0076] The molar ratio of Compound 4a to the condensing agent is 1:(1 - 3); for example, 1:1, 1:2, 1:3 and / or,

[0077] In Method 1, Method 2, Method 3, and step (4) of Method 4, the reaction time is independently 5 - 10 h; for example, 5, 6, 7, 8, 9, 10 h; and / or,

[0078] In Method 4,

[0079] In step (1), the mass ratio of Compound 4 to the cyclization catalyst is 1:(40 - 60); for example, 1:40, 1:50, 1:60; and / or,

[0080] In step (1), the temperature of the heating reaction is 120 - 160 °C; for example, 120, 130, 140, 150, 160 °C; and / or,

[0081] In step (1), the time of the heating reaction is 4 - 5 h; for example, 4, 5 h; and / or,

[0082] In step (2), after adding the alkali solution, the pH value of the reaction system is 6 - 7; and / or,

[0083] The reaction time of step (2) is 2 - 4 h; for example, 2, 3, 4 h; and / or,

[0084] Step (3), the molar ratio of compound 4a to R 4a1 -X is 1:(1 - 2); and / or,

[0085] Step (3), the molar ratio of compound 4a to the nucleophile is 1:(1 - 2); and / or,

[0086] Step (3), the molar volume ratio of compound 4a to the solvent is 1 mmol:(20 - 60) mL; and / or,

[0087] Step (3), the molar ratio of compound 4a to the acid-binding agent is 1:(6 - 9); such as 1:6, 1:7, 1:8, 1:9; and / or,

[0088] The reaction time of step (3) is 2 - 8 h; such as 2, 3, 4, 5, 6, 7, 8 h.

[0089] The preparation methods of compound 1, compound 2, and compound 3 are as follows: Dissolve ethyl 3-aminocrotonate in an acid solvent (preferably glacial acetic acid), heat (preferably at 55 - 65 °C) for a period of time (preferably 15 - 20 min), and then dissolve one of the dihydroxybenzaldehydes as in an acid solvent (preferably glacial acetic acid), and slowly drop it into the above reaction system for reaction (preferably for more than 6 hours) to obtain compound 1, compound 2, and compound 3;

[0090] Preferably, the molar ratio of ethyl 3-aminocrotonate to the dihydroxybenzaldehyde is 2 - 3:1.

[0091] The preparation method of compound 4 is as follows: React 7-amino-7-hydroxycoumarin with dimethyl acetylenedicarboxylate in an alcohol solvent under the action of a metal catalyst Mg at room temperature to obtain compound 4.

[0092] The third object of the present invention is to provide an application of a pyridone-type coumarin derivative as described in the first object of the present invention or a pyridone-type coumarin derivative prepared by the method as described in the second object of the present invention in at least one of anti-inflammatory, antiviral, antibacterial, or anticancer.

[0093] In the application described in the present invention, preferably,

[0094] The application of the pyridone-type coumarin derivative in inhibiting KYSE-70 and / or KYSE-150 cells.

[0095] In the ranges disclosed in the present invention, the endpoints and any values of the ranges are not limited to the exact 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 each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. In the following text, in principle, various technical solutions can be combined with each other to obtain new technical solutions, and this should also be regarded as specifically disclosed herein.

[0096] Compared with the prior art, the present invention has at least the following advantages:

[0097] The pyridone-coumarin derivative of the present invention has a significant inhibitory effect on KYSE-70 and KYSE-150 cells and has very good application value. Specific embodiments

[0098] The present invention will be specifically described below in conjunction with specific embodiments. It is necessary to point out here that the following embodiments are only used for further illustration of the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art to the present invention based on the content of the present invention still fall within the protection scope of the present invention.

[0099] In addition, it should be noted that, among the various specific technical features described in the following specific embodiments, without contradiction, they can be combined in any

[0100] suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0101] In addition, any combination can be made among various different embodiments of the present invention as long as it does not violate the idea of the present invention. The technical solutions formed thereby belong to a part of the original disclosure content of this specification and also fall within the protection scope of the present invention.

[0102] If there is no special limitation on the raw materials used in the examples and comparative examples, they are all disclosed in the prior art. For example, they can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0103] Example 1

[0104] Synthesis of Compound 1

[0105]

[0106] Ethyl 3-aminocrotonate (4 mmol) was dissolved in 1.1 mL of glacial acetic acid and placed in a 25 mL round-bottom flask equipped with a stirrer and a condenser. The mixture was heated to 60 °C and the reaction was started. After 15 minutes, 2,3-dihydroxybenzaldehyde (2 mmol) in 0.55 mL of glacial acetic acid was slowly added dropwise to the reaction system. The reaction was stopped after 6 hours. After the reaction was completed, the round-bottom flask was allowed to stand at room temperature for 12 hours, and a large amount of solid was observed to precipitate. The solid was collected by suction filtration to obtain the crude product, which was then separated and purified by column chromatography to finally obtain the target parent compound 1 (ethyl 2,4-dimethyl-7-hydroxy-3-formylcoumarin[4,3-b]pyridine).

[0107] The yield was 32.25%, and the melting point was 203 - 205 °C. 1 H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 7.18 (m, 1H), 4.49 (q, J = 7.1 Hz, 2H), 2.80 (s, 3H), 2.68 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 167.98, 160.94, 159.14, 152.61, 150.14, 143.23, 140.52, 131.83, 124.81, 119.57, 118.22, 116.75, 113.57, 62.13, 23.80, 19.49, 14.19.

[0108] Synthesis of Compound 1a

[0109]

[0110] 82.8 mg (3 mmol) of (E)-2-methyl-2-pentenoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 58.7 mg of ethyl 2,4-dimethyl-7-hydroxy-3-formylcoumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.365 g of the purified product, a milky white solid, with a yield of 65.34% and a melting point of 108 - 110 °C.

[0111] Example 2

[0112] Synthesis of Compound 1b

[0113]

[0114] 175.2 mg (3 mmol) of 3,5-dibromobenzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 59.0 mg of 2,4-dimethyl-7-hydroxy-3-ethyl carboxylate coumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.265 g of the purified product as a milky white solid, with a yield of 56.70% and a melting point of 194 - 197 °C.

[0115] Example 3

[0116] Synthesis of Compound 1c

[0117]

[0118] 132.8 mg (3 mmol) of p-bromo-o-tolylbenzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 58.7 mg of 2,4-dimethyl-7-hydroxy-3-ethyl carboxylate coumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.568 g of the purified product as a milky white solid, with a yield of 68.50% and a melting point of 137 - 138 °C.

[0119] Example 4

[0120] Synthesis of Compound 1d

[0121]

[0122] Using ultrasonic assistance, 111.1 mg (3 mmol) of benzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 86.2 mg of 2,4-dimethyl-7-hydroxy-3-ethyl carboxylate coumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.668 g of the purified product as a milky white solid, with a yield of 75.50% and a melting point of 140 - 143 °C.

[0123] Example 5

[0124] Synthesis of Compound 1e

[0125]

[0126] Using ultrasonic assistance, 128.2 mg (3 mmol) of 3,3-dimethylacrylic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 58.8 mg of 2,4-dimethyl-7-hydroxy-3-ethyl carboxylate coumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.628 g of the purified product as a milky white solid, with a yield of 62.50% and a melting point of 127 - 129 °C.

[0127] Example 6

[0128]

[0129] Ethyl 3-aminocrotonate (4 mmol) was dissolved in 1.1 mL of glacial acetic acid and placed in a 25 mL round-bottom flask equipped with a stirrer and a condenser. The temperature was heated to 60 °C and the reaction was started. After 15 minutes, 2,4-dihydroxybenzaldehyde (2 mmol) in 0.55 mL of glacial acetic acid was slowly added dropwise to the reaction system. The reaction was stopped after 6 hours. After the reaction was completed, the round-bottom flask was left to stand at room temperature for 12 hours, and a large amount of solid was observed to precipitate. The solid was collected by suction filtration to obtain the crude product, which was then separated and purified by column chromatography to finally obtain the target parent compound 2.

[0130] The yield was 36.74% and the melting point was 195 °C. 1¹H NMR (600 MHz, Methanol-d₄) δ 8.32 (d, J = 8.7 Hz, 1H), 6.81 (dd, J = 2.3, 8.7 Hz, 1H), 6.65 (d, J = 2.3 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 2.70 (s, 3H), 2.59 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, Methanol-d₄) δ 168.12, 161.97, 160.64, 160.14, 154.23, 152.75, 149.54, 130.36, 126.51, 113.00, 111.72, 110.89, 101.68, 61.73, 22.42, 18.12, 13.05.

[0131] Synthesis of Compound 2a

[0132]

[0133] 62.5 mg (3 mmol) of (E)-2-methyl-2-butenoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 58.4 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinopyridine[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.586 g of the purified product as a milky white solid, with a yield of 58.60% and a melting point of 134 - 135 °C.

[0134] Example 7

[0135] Synthesis of Compound 2b

[0136]

[0137] 135.5 mg (3 mmol) of p-bromo-o-toluic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 59.0 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridinecarboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.686 g of the purified product as a milky white solid, with a yield of 68.60% and a melting point of 149 - 152 °C.

[0138] Example 8

[0139] Synthesis of Compound 2c

[0140]

[0141] 71.7 mg (3 mmol) of p-bromo-o-toluic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 58.0 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridinecarboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.656 g of the purified product as a milky white solid, with a yield of 64.20% and a melting point of 103 - 107 °C.

[0142] Example 9

[0143] Synthesis of Compound 2d

[0144]

[0145] Using ultrasonic assistance, 173.9 mg (3 mmol) of 3,5-dibromobenzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 58.5 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinylpyridine[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The reaction progress was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.556 g of the purified product as a milky white solid, with a yield of 54.35% and a melting point of 203-204 °C.

[0146] Example 10

[0147] Synthesis of Compound 2e

[0148]

[0149] Using ultrasonic assistance, 129.3 mg (3 mmol) of 2,4-dichloro-5-fluorobenzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 57.5 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinylpyridine[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The reaction progress was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.586 g of the purified product as a milky white solid, with a yield of 53.65% and a melting point of 143-146 °C.

[0150] Example 11

[0151] Synthesis of Compound 2f

[0152]

[0153] 84 mg (3 mmol) of 3-chloro-4-methylbenzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 59.8 mg of ethyl 2,4-dimethyl-8-hydroxycoumarino[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.653 g of the purified product as a milky white solid, with a yield of 62.75% and a melting point of 142 - 143 °C.

[0154] Example 12

[0155] Synthesis of Compound 2g

[0156]

[0157] 120.4 mg (3 mmol) of 4-trifluoromethylbenzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 65.0 mg of ethyl 2,4-dimethyl-8-hydroxycoumarino[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.723 g of the purified product as a milky white solid, with a yield of 72.75% and a melting point of 198 - 200 °C.

[0158] Example 13

[0159] Synthesis of Compound 2h

[0160]

[0161] Using ultrasonic assistance, 84.5 mg (3 mmol) of benzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 65.4 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridinecarboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.756 g of the purified product as a milky white solid, with a yield of 76.35% and a melting point of 197 - 198 °C.

[0162] Example 14

[0163] Synthesis of Compound 2i

[0164]

[0165] Using ultrasonic assistance, 125.6 mg (3 mmol) of 2-trifluoromethylbenzoic acid, 93 mg of the condensing agent EDC, and 9.8 mg of the catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 58.6 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridinecarboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored using TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out using column chromatography (wet loading) to obtain 0.727 g of the purified product as a milky white solid, with a yield of 72.45% and a melting point of 137 - 139 °C.

[0166] Example 15

[0167] Synthesis of Compound 2j

[0168]

[0169] Under ultrasonic assistance, 128.5 mg (3 mmol) of p-trifluoromethylsalicylic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 58.5 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.625 g of the purified product as a milky white solid, with a yield of 62.35% and a melting point of 229 - 231 °C.

[0170] Example 16

[0171] Synthesis of Compound 2k

[0172]

[0173] Under ultrasonic assistance, 84.0 mg (3 mmol) of 3-bromo-4-trifluoromethylbenzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 59.8 mg of ethyl 2,4-dimethyl-8-hydroxy-3-coumarinyl[4,3-b]pyridine-3-carboxylate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.665 g of the purified product as a milky white solid, with a yield of 65.35% and a melting point of 195 - 197 °C.

[0174] Example 17

[0175]

[0176] Ethyl 3-aminocrotonate (4 mmol) was dissolved in 1.1 mL of glacial acetic acid and placed in a 25 mL round-bottom flask equipped with a stirrer and a condenser. The temperature was heated to 60 °C and the reaction was started. After 15 minutes, 2,5-dihydroxybenzaldehyde (2 mmol) in 0.55 mL of glacial acetic acid was slowly added dropwise to the reaction system. The reaction was stopped after 6 hours. After the reaction was completed, the round-bottom flask was left to stand at room temperature for 12 hours, and a large amount of solid was observed to precipitate. The solid was collected by suction filtration to obtain the crude product, which was then separated and purified by column chromatography to finally obtain the target parent compound 3.

[0177] The yield was 22.35% and the melting point was 248.5 °C.1 1H NMR (400 MHz, DMSO-d6) δ 9.68 (d, J = 6.9 Hz, 1H), 7.74 (m, 1H), 7.19 (s, 1H), 7.00 (dd, J = 2.9, 5.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 2.60 (s, 3H), 2.54 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 168.00, 167.37, 159.47, 154.15, 151.52, 149.00, 145.50, 131.10, 120.67, 119.04, 117.45, 113.69, 109.15, 62.00, 23.43, 19.01, 13.98.

[0178] Synthesis of Compound 3a

[0179]

[0180] 98.5 mg (3 mmol) of 4-trifluoromethylbenzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane using ultrasonic assistance. The temperature was raised to 25 °C, and 58.8 mg of ethyl 2,4-dimethyl-9-hydroxy-3-carboxylate coumarin[4,3-b]pyridine dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.685 g of the purified product as a milky white solid, with a yield of 68.25% and a melting point of 170 - 171 °C.

[0181] Example 18

[0182] Synthesis of Compound 3b

[0183]

[0184] Using ultrasonic assistance, 80.3 mg (3 mmol) of benzoic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 57.1 mg of ethyl 2,4-dimethyl-9-hydroxy-3-coumarinylnicotinate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.727 g of the purified product as a milky white solid, with a yield of 69.76% and a melting point of 195 - 198 °C.

[0185] Example 19

[0186] Synthesis of Compound 3c

[0187]

[0188] Using ultrasonic assistance, 52.7 mg (3 mmol) of 2-thiophenecarboxylic acid, 93 mg of condensing agent EDC, and 9.8 mg of catalyst DMAP were dissolved in 20 mL of anhydrous dichloromethane. The temperature was raised to 25 °C, and 57.3 mg of ethyl 2,4-dimethyl-9-hydroxy-3-coumarinylnicotinate dissolved in 10 mL of anhydrous dichloromethane was added dropwise to the round-bottom flask containing the previous step. The reaction was stirred at 25 °C for 6 hours. The progress of the reaction was monitored by TLC thin-layer chromatography. During the reaction, the solution changed from colorless to milky white and a solid precipitated. The reaction was stopped, and gradient elution was carried out by column chromatography (wet loading) to obtain 0.538 g of the purified product as a milky white solid, with a yield of 59.85% and a melting point of 180 - 182 °C.

[0189] Structure Characterization

[0190] Example 20

[0191] Synthesis of 7-hydroxy-4-aminocoumarin (This compound has a known compound structure)

[0192]

[0193] At room temperature, resorcinol (22.84 g, 0.2 mol), cyanoacetic acid (19.33 g, 0.2 mol), zinc chloride (13.95 g, 0.1 mmol) and ether (50 mL) were successively placed in a dry three-necked round-bottom flask. A condenser and a drying tube filled with anhydrous calcium chloride were installed. Hydrogen chloride gas and nitrogen were continuously introduced, and the reaction was vigorously stirred for 5 h. A large amount of white solid precipitate appeared in the three-necked round-bottom flask. The reaction was stopped, quenched with distilled water, and the white solid was filtered by suction and dried. The yield of the target product was 23.7% (8.41 g), and the melting point was 272.2 - 273 °C

[0194] Example 21

[0195] Synthesis of Compound 4

[0196]

[0197] 7-Amino-7-hydroxycoumarin (200 mg, 1.13 mmol) was weighed into a 50 mL single-necked flask, and then dimethyl acetylenedicarboxylate (729.02 mg, 5.11 mmol) was added. To promote the reaction process, a metal catalyst Mg (2.9 mg, 0.12 mmol) was added, and the reaction was carried out at room temperature for 4 h using anhydrous methanol (10 mL) as the solvent. As the reaction proceeded, the solution changed from white to transparent reddish-brown, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was filtered, and after the filtrate was concentrated, the crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4

[0198] Example 22

[0199] Synthesis of Compound 4a

[0200]

[0201] Compound 4 (100 mg, 0.31 mmol) was taken in a 50 mL round-bottom flask, and polyphosphoric acid (PPA, 5 g) was added. After evacuation and introduction of nitrogen three times, the temperature was raised to 130 °C and the reaction was carried out for 4 - 5 h. The reaction mixture was cooled to 5 °C, and the mixture was slowly transferred to an excess of saturated sodium carbonate solution at 10 °C. The solid was filtered and washed with 20 mL of water. Then the solid was dried to obtain compound 4a

[0202] Example 23

[0203] Synthesis of Compound 4b

[0204]

[0205] Compound 4a (287 mg, 1.0 mmol) was added to a 10% NaOH solution to adjust the pH of the reaction system to about 7. The mixture was stirred at room temperature for 2 h, and a white solid was observed to precipitate. The solid was filtered and washed with water. Then the solid was dried to obtain white solid compound 4b.

[0206] Example 24

[0207] Synthesis of Compound 4c

[0208]

[0209] N-Boc-4-chloropiperidine (115.35 mg, 0.53 mmol) was weighed into a 50 mL round-bottom flask, and KI (iodide ion is a good nucleophile and also a good leaving group. Therefore, a very small amount of potassium iodide is added during the nucleophilic substitution of alkanes to make the leaving group of the original reagent easily attacked by the iodide ion and lost to form an iodoalkane. The iodine in the iodoalkane is then easily attacked by other nucleophiles and lost, so the reaction rate can be accelerated) (87.15 mg, 0.53 mmol) was added. Using DMF (10 mL) as the solvent, after reacting at room temperature for 30 min, compound 4a (100 mg, 0.35 mmol) and potassium carbonate (a base scavenger, acid is generated during the reaction and the addition of base neutralizes it to promote the forward reaction) (370 mg) were added and the reaction continued for 3 h. The reaction process was monitored by TLC. When the reaction was completed, 10 mL of distilled water was added, and then the mixture was extracted with ethyl acetate three times. The organic phase was concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4c.

[0210] Example 25

[0211] Synthesis of Compound 4d

[0212] Cyclopropanecarboxylic acid (86 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) (EDC is a condensing agent in the esterification reaction, and dmap acts as a strong basic catalyst to promote the esterification reaction) were added to a 50 mL round-bottom flask, and dichloromethane (10 mL) was added and stirred at room temperature for 20 min. Then compound 4a (143.6 mg, 0.50 mmol) was weighed and added to the reaction flask, and the mixture was stirred and reacted for 6 h, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4d.

[0213]

[0214] Example 26

[0215] Synthesis of Compound 4e

[0216]

[0217] Weigh 4-bromothiophene-2-acetic acid (221.1 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) and add them into a 50 mL round-bottom flask. Add dichloromethane (10 mL) and stir at room temperature for 20 min. Then weigh 4a (143.6 mg, 0.50 mmol) and add it into the reaction flask, stir and react for 6 h, and detect the reaction by TLC. After the reaction is completed, evaporate the reaction solution under reduced pressure, and purify the crude product by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4e.

[0218] Example 27

[0219] Synthesis of Compound 4f

[0220]

[0221] Weigh 4-bromothiophene-3-carboxylic acid (207 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) and add them into a 50 mL round-bottom flask. Add dichloromethane (10 mL) and stir at room temperature for 20 min. Then weigh 4a (143.6 mg, 0.50 mmol) and add it into the reaction flask, stir and react for 6 h, and detect the reaction by TLC. After the reaction is completed, evaporate the reaction solution under reduced pressure, and purify the crude product by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4f.

[0222] Example 28

[0223] Synthesis of Compound 4g

[0224]

[0225] Weigh 3-chlorothiophene-2-carboxylic acid (162.6 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) and add them into a 50 mL round-bottom flask. Add dichloromethane (10 mL) and stir at room temperature for 20 min. Then weigh 4a (143.6 mg, 0.50 mmol) and add it into the reaction flask, stir and react for 6 h, and detect the reaction by TLC. After the reaction is completed, evaporate the reaction solution under reduced pressure. The crude product is purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4g.

[0226] Example 29

[0227] Synthesis of Compound 4h

[0228]

[0229] Weigh 4-bromothiophene-2-carboxylic acid (207 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) and add them into a 50 mL round-bottom flask. Add dichloromethane (10 mL) and stir at room temperature for 20 min. Then weigh 4a (143.6 mg, 0.50 mmol) and add it into the reaction flask, stir and react for 6 h, and detect the reaction by TLC. After the reaction is completed, evaporate the reaction solution under reduced pressure. The crude product is purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4h.

[0230] Example 30

[0231] Synthesis of Compound 4i

[0232]

[0233] Weigh 5-methyl-1,2-oxazole-4-carboxylic acid (127 mg, 1.00 mmol), EDC (156.1 mg, 1.00 mmol) and DMAP (18.8 mg, 0.15 mmol) and add them into a 50 mL round-bottom flask. Add dichloromethane (10 mL) and stir at room temperature for 20 min. Then weigh 4a (143.6 mg, 0.50 mmol) and add it into the reaction flask, stir and react for 6 h, and detect the reaction by TLC. After the reaction is completed, evaporate the reaction solution under reduced pressure. The crude product is purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1 / 50 - 20, V / V) to obtain the target compound 4i.

[0234] The structural characterization results of the target compounds prepared in the above Examples 1 - 30 are as follows:

[0235] 1. Compound 1a

[0236] 1H NMR (600 MHz, Chloroform-d) δ 8.49 (t, J = 4.8 Hz, 1H), 7.35 (d, J = 5.0 Hz, 2H), 4.48 (q, J = 7.2 Hz, 2H), 2.77 (s, 3H), 2.68 (s, 3H), 1.92 (d, J = 7.2 Hz, 3H), 1.85–1.80 (m, 5H), 1.44 (t, J = 7.1 Hz, 3H).

[0237] 13C NMR (151 MHz, Chloroform-d) δ 173.10, 168.14, 165.91, 160.88, 159.21, 152.29, 150.16, 144.98, 140.86, 140.04, 138.44, 132.10, 128.10, 127.64, 126.08, 124.30, 122.89, 120.66, 114.02, 62.25, 19.57, 14.95, 14.76, 14.34, 12.45, 11.83.

[0238] 2. Compound 1b

[0239] 1H NMR (600 MHz, Chloroform-d) δ 8.10 (d, J = 8.1 Hz, 1H), 7.24 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.79 (s, 3H), 2.68 (s, 3H), 1.44 (d, J = 14.3 Hz, 3H).

[0240] 13C NMR (151 MHz, Chloroform-d) δ 168.00, 160.95, 159.16, 152.62, 150.12, 143.24, 140.52, 131.83, 124.81, 119.59, 118.22, 116.74, 113.56, 62.14, 23.82, 19.50, 14.20.

[0241] 3. Compound 1c

[0242] 1H NMR (600 MHz, Chloroform-d) δ 8.54 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 7.53–7.48 (m, 2H), 7.46–7.39 (m, 2H), 4.49 (q, J = 7.1 Hz, 2H), 2.77 (s, 3H), 2.69 (d, J = 8.3 Hz, 6H), 1.44 (t, J = 7.1 Hz, 3H).

[0243] 13C NMR (151 MHz, Chloroform-d) δ 168.06, 164.54, 160.96, 159.01, 152.16, 150.20, 144.84, 143.79, 137.93, 134.98, 133.07, 132.19, 129.45, 129.29, 128.09, 126.95, 125.92, 124.38, 123.31, 120.81, 114.05, 62.26, 29.85, 23.94, 22.02, 21.86, 19.54, 14.32.

[0244] 4. Compound 1d

[0245] 1H NMR (600 MHz, Chloroform-d) δ 8.55 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 7.3 Hz, 2H), 8.12 (d, J = 8.2 Hz, 2H), 7.68 (t, J = 8.1 Hz, 1H), 7.65–7.59 (m, 1H), 7.57–7.52 (m, 2H), 7.51–7.45 (m, 3H), 7.41 (d, J = 9.3 Hz, 1H), 4.52–4.45 (m, 2H), 2.77 (s, 3H), 2.70 (s, 3H), 1.44 (d, J = 14.3 Hz, 3H).

[0246] 13C NMR (151 MHz, Chloroform-d) δ 168.10, 164.61, 160.91, 159.07, 152.21, 150.18, 144.94, 138.14, 134.07, 133.92, 132.15, 130.70, 130.34, 129.37, 128.85 (d, J = 13.7 Hz), 128.63, 125.98, 124.36, 123.23, 120.80, 114.05, 62.25, 23.94, 19.55, 14.32.

[0247] 5. Compound 1e

[0248] 1H NMR (600 MHz, Chloroform-d) δ 8.48 (t, J = 4.8 Hz, 1H), 7.34 (s, 2H), 4.48 (q, J = 7.2 Hz, 2H), 2.78 (s, 3H), 2.68 (s, 3H), 2.25 (s, 3H), 2.03 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H).

[0249] 13C NMR (151 MHz, Chloroform-d) δ 168.13, 164.10, 161.41, 160.83, 159.23, 152.29, 150.11, 144.97, 137.97, 132.04, 126.14, 124.25, 122.82, 120.61, 115.44, 114.67, 114.00, 62.21, 27.92, 23.92, 20.98, 20.81, 19.55, 14.32.

[0250] 6. Compound 2a

[0251] 1H NMR (600 MHz, Chloroform-d) δ 7.17 (d, J = 7.2 Hz, 1H), 4.48 (q, J = 7.2 Hz, 1H), 2.79 (s, 2H), 2.68 (s, 2H), 1.91 (d, J = 7.2 Hz, 2H), 1.85–1.80 (m, 1H), 1.44 (t, J = 7.2 Hz, 2H).

[0252] 13C NMR (151 MHz, Chloroform-d) δ 168.18, 165.97, 160.90, 160.19, 154.40, 153.26, 152.16, 150.15, 140.68, 131.79, 127.93, 126.71, 118.81, 116.76, 113.48, 110.26, 62.21, 23.93, 19.54, 14.91, 14.32, 12.31.

[0253] 7. Compound 2b

[0254] 1H NMR (600 MHz, Chloroform-d) δ 8.67 (d, J = 9.4 Hz, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.53–7.47 (m, 2H), 4.52–4.46 (m, 2H), 2.80 (s, 3H), 2.68 (d, J = 15.9 Hz, 6H), 1.44 (t, J = 7.2 Hz, 3H), 1.25 (s, 1H).

[0255] 13C NMR (151 MHz, Chloroform-d) δ 168.13, 164.47, 160.97, 160.06, 153.80, 153.29, 152.02, 150.19, 143.96, 135.17, 132.88, 131.93, 129.49, 128.29, 126.86 (d, J = 17.0 Hz), 118.73, 117.15, 113.57, 110.35, 62.24, 23.94, 21.99, 19.53, 14.32.

[0256] 8. Compound 2c

[0257] 1H NMR (600 MHz, Chloroform-d) δ 8.61 (d, J = 9.6 Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 2.79 (s, 3H), 2.68 (s, 3H), 2.30 (p, J = 7.7 Hz, 2H), 1.97 (s, 3H), 1.46–1.41 (m, 4H), 1.27 (d, J = 17.9 Hz, 4H), 1.13 (t, J = 7.5 Hz, 3H).

[0258] 9. Compound 2d

[0259] 1H NMR (600 MHz, Chloroform-d) δ 8.69 (d, J = 8.7 Hz, 1H), 8.28 (s, 2H), 7.96 (s, 1H), 4.50 (q, J = 7.2, 6.1 Hz, 2H), 2.81 (s, 3H), 2.69 (s, 3H), 1.45 (d, J = 14.3 Hz, 3H).

[0260] 13C NMR (151 MHz, Chloroform-d) δ 168.18, 166.15, 160.89, 160.19, 154.42, 153.27, 152.16, 150.15, 147.38, 131.78, 126.70, 126.43, 118.82, 116.75, 113.48, 110.27, 62.21, 29.84, 23.93, 22.51, 19.53, 14.32, 13.07, 12.48.

[0261] 10. Compound 2e

[0262] 1H NMR (600 MHz, Chloroform-d) δ 8.68 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 6.3 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.80 (s, 3H), 2.70 (s, 3H), 1.45 (d, J = 6.9 Hz, 3H).

[0263] 13C NMR (151 MHz, Chloroform-d) δ 168.08, 162.22, 161.02, 159.96, 153.33 (d, J = 19.5 Hz), 151.89, 150.21, 139.42, 132.27, 132.08, 127.08, 123.49, 118.36, 117.49, 113.65, 110.14, 62.26, 29.84, 23.93, 19.52, 14.32.

[0264] 11. Compound 2f

[0265] 1H NMR (600 MHz, Chloroform-d) δ 8.67 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 4.50 (q, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.69 (s, 3H), 2.49 (s, 3H), 1.45 (d, J = 14.3 Hz, 3H).

[0266] 13C NMR (151 MHz, Chloroform-d) δ 168.13, 163.64, 160.97, 160.07, 153.87, 153.30, 152.03, 150.20, 143.01, 135.09, 131.93, 131.37, 130.94, 128.58, 128.28, 126.93, 118.62, 117.20, 113.60, 110.26, 62.23, 29.84, 23.93, 20.66, 19.51, 14.33.

[0267] 12. Compound 2g

[0268] 1H NMR (600 MHz, Chloroform-d) δ 8.67 (d, J = 8.1 Hz, 1H), 8.33 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H), 4.48 (q, J = 7.1 Hz, 2H), 2.79 (s, 3H), 2.68 (s, 3H), 1.43 (d, J = 14.3 Hz, 3H).

[0269] 13C NMR (151 MHz, Chloroform-d) δ 168.09, 163.52, 161.02, 160.00, 153.62, 153.30, 151.94, 150.22, 132.35, 132.01, 130.87, 130.69, 127.04, 125.92, 122.72, 118.49, 117.40, 113.64, 110.21, 62.26, 29.84, 23.93, 19.51, 14.32.

[0270] 13. Compound 2h

[0271] 1H NMR (600 MHz, Chloroform-d) δ 8.67 (d, J = 9.3 Hz, 1H), 8.23 (d, J = 9.6 Hz, 2H), 8.12 (d, J = 9.6 Hz, 1H), 7.67 (s, 1H), 7.64–7.59 (m, 1H), 7.54 (t, J = 7.8 Hz, 2H), 7.48 (t, J = 7.8 Hz, 1H), 4.49 (q, J = 7.1 Hz, 2H), 2.80 (s, 3H), 2.69 (s, 3H), 1.45 (d, J = 14.3 Hz, 3H).

[0272] 14. Compound 2i

[0273] 1H NMR (600 MHz, Chloroform-d) δ 8.65 (d, J = 9.4 Hz, 1H), 7.98 (d, J = 4.1 Hz, 1H), 7.85–7.80 (m, 1H), 7.72–7.67 (m, 2H), 4.46 (q, J = 7.2 Hz, 2H), 2.77 (s, 3H), 2.66 (s, 3H), 1.41 (d, J = 14.3 Hz, 3H).

[0274] 13C NMR (151 MHz, Chloroform-d) δ 168.11, 164.64, 160.99, 159.98, 153.50, 153.28, 151.95, 150.19, 132.30, 132.15, 131.99, 130.99, 129.90, 127.71–126.82 (m), 118.27, 117.43, 113.64, 110.01, 62.24, 29.84, 23.93, 19.51, 14.32.

[0275] 15. Compound 2j

[0276] 1H NMR (600 MHz, Chloroform-d) δ 10.46 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 7.23 (d, J = 8.5 Hz, 2H), 4.49 (q, J = 7.1 Hz, 2H), 2.80 (s, 3H), 2.69 (s, 3H), 1.44 (d, J = 14.3 Hz, 3H).

[0277] 13C NMR (151 MHz, Chloroform-d) δ 168.04, 167.61, 162.40, 161.11, 159.86, 153.30, 152.65, 151.78, 150.27, 132.18, 131.45, 127.22, 118.36, 117.85, 116.16, 115.62, 114.26, 113.76, 110.26, 62.30, 31.39, 29.85, 23.94, 19.52, 14.33.

[0278] 16. Compound 2k

[0279] 1H NMR (600 MHz, Chloroform-d) δ 8.70 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.26 (dd, J = 11.2, 2.7 Hz, 3H), 4.49 (q, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.70 (s, 3H), 1.45 (d, J = 14.3 Hz, 3H).

[0280] 13C NMR (151 MHz, Chloroform-d) δ 168.06, 162.34, 161.05, 159.93, 153.31 (d, J = 6.8 Hz), 151.86, 150.25, 136.56, 133.52, 132.08, 129.09, 128.35, 127.14, 123.44, 120.77, 118.34, 117.57, 113.69, 110.15, 62.28, 23.94, 19.53, 14.33.

[0281] 17. Compound 3a

[0282] 1H NMR (600 MHz, Chloroform-d) δ 8.46 (s, 1H), 8.36 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 4.0 Hz, 2H), 4.49 (q, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.67 (s, 3H), 1.44 (d, J = 14.3 Hz, 3H).

[0283] 13C NMR (151 MHz, Chloroform-d) δ 168.02, 164.17, 160.95, 159.95, 151.75, 150.53, 150.24, 147.23, 135.32, 132.58, 132.40, 130.80, 125.95 (d, J = 21.4 Hz), 118.33, 118.10, 114.05, 62.29, 23.85, 19.53, 14.31.

[0284] 18. Compound 3b

[0285] 1H NMR (600 MHz, Chloroform-d) δ 8.45 (s, 1H), 8.24 (d, J = 9.9 Hz, 2H), 7.67 (t, J = 7.5 Hz, 1H), 7.56–7.51 (m, 2H), 7.41 (t, J = 8.8 Hz, 2H), 4.49 (q, J = 7.2 Hz, 2H), 2.81 (s, 3H), 2.66 (s, 3H), 1.44 (d, J = 14.4 Hz, 3H).

[0286] 13C NMR (151 MHz, Chloroform-d) δ 168.07, 165.36, 160.89, 160.06, 151.87, 150.36, 150.18, 147.57, 133.99, 133.82, 132.28, 130.35 (d, J = 13.3 Hz), 129.30, 128.81, 128.61, 126.28, 120.18, 118.45, 117.95, 113.98, 62.25, 29.84, 23.84, 19.53, 14.31.

[0287] 19. Compound 3c

[0288] 1H NMR (600 MHz, Chloroform-d) δ 8.46 (d, J = 2.8 Hz, 1H), 8.02 (d, J = 3.8 Hz, 1H), 7.70 (d, J = 5.0 Hz, 1H), 7.45–7.38 (m, 2H), 7.23–7.18 (m, 1H), 4.48 (q, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.67 (s, 3H), 1.44 (d, J = 14.3 Hz, 3H).

[0289] 13C NMR (151 MHz, Chloroform-d) δ 168.06, 160.89, 160.73, 160.02, 151.83, 150.39, 150.16, 147.15, 135.16, 134.03, 132.52, 132.29, 126.22, 120.16, 118.44, 117.94, 113.97, 62.26, 29.84, 19.54, 14.32.

[0290] 21. Compound 4

[0291] White solid, yield 80.6%. 1 1H NMR (600 MHz, Methanol-d4) δ 7.87 (d, J = 8.9 Hz, 1H), 6.97 (dd, J = 8.9, 2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.78 (s, 1H), 5.31 (s, 1H), 3.79 (s, 3H), 3.70 (s, 3H). 1313C NMR (150 MHz, Methanol-d4) δ 166.59, 164.90, 163.29, 161.28, 158.55, 156.53, 149.79, 125.32, 118.07, 113.71, 111.22, 104.88, 83.79, 53.74, 52.58.

[0292] Compound 4a

[0293] White solid, yield 10.8%. 1 1H NMR (600 MHz, Methanol-d4) δ 7.42 (dd, J = 8.5, 2.1 Hz, 1H), 6.62 (dt, J = 8.7, 2.3 Hz, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.91 (s, 1H), 2.35 (d, J = 2.1 Hz, 3H). 13 13C NMR (150 MHz, Methanol-d4) δ 176.78, 164.88, 163.56, 161.52, 154.24, 147.17, 137.51, 126.41, 111.44, 110.58, 110.07, 102.40, 102.08, 53.09.

[0294] 23(2-Formyl[3,4]pyridine)-7-hydroxycoumarin (Compound 4b)

[0295] White solid, yield 63.4%. 1 1H NMR (600 MHz, Methanol-d4) δ 7.46 (d, J = 8.6 Hz, 1H), 6.66 (dt, J = 8.7, 2.1 Hz, 1H), 6.54–6.50 (m, 1H), 5.95 (s, 1H). 13 13C NMR (150 MHz, Methanol-d4) δ 177.31, 164.88, 164.52, 161.52, 154.24, 147.31, 137.57, 126.41, 112.42, 110.58, 110.00, 102.40, 102.08.

[0296] 24. (2-Methyl formate[3,4]pyridine)-7-(N-Boc-4-piperidylamino)coumarin (Compound 4c)

[0297] White solid, yield 52.3%. 11H NMR (600 MHz, Methanol-d4) δ 7.43 (dd, J = 8.7, 2.0 Hz, 1H), 6.67–6.59 (m, 1H), 6.48 (d, J = 2.4 Hz, 1H), 5.92 (s, 1H), 4.59 (s, 1H), 4.23 (p, J = 5.5, 4.8 Hz, 2H), 3.70–3.67 (m, 2H), 3.29–3.28 (m, 3H), 2.35 (s, 2H), 2.03–2.00 (m, 2H), 1.71–1.68 (m, 2H), 1.43 (d, J = 2.0 Hz, 9H). 13 13C NMR (150 MHz, Methanol-d4) δ 176.78, 169.52, 164.87, 163.56, 160.67, 153.85, 147.16, 137.51, 126.60, 111.94, 111.56, 111.44, 104.10, 102.40, 73.83, 53.09, 49.07, 44.15, 31.15, 30.61, 30.59.

[0298] 25. Methyl (2-(pyrido[3,4-b]pyridin-2-yl))-7-(cyclopropanecarboxylate)coumarin (Compound 4d)

[0299] White solid, yield 52.3%. 1 1H NMR (600 MHz, DMSO-d6) δ 8.02 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.14 (dd, J = 8.7, 2.3 Hz, 1H), 5.92 (s, 1H), 1.92 (tt, J = 7.9, 4.7 Hz, 1H), 1.08 (dt, J = 7.3, 2.7 Hz, 2H), 1.07–1.03 (m, 2H). 13 13C NMR (150 MHz, DMSO-d6) δ 176.78, 172.92, 164.87, 163.56, 154.24, 153.71, 147.07, 137.51, 126.59, 116.82, 111.98, 111.44, 108.50, 102.40, 53.09, 15.14, 9.26.

[0300] 26. Methyl (2-(pyrido[3,4-b]pyridin-2-yl))-7-(4-bromothiophene-2-carboxylate)coumarin (4e)

[0301] Yellow solid, yield 52.3%. 11H NMR (600 MHz, Chloroform-d) δ 7.64 (d, J = 1.4 Hz, 1H), 7.51 (d, J = 1.4 Hz, 1H), 7.34 (d, J = 1.4 Hz, 1H), 7.23 (d, J = 1.4 Hz, 1H), 7.20 (s, 2H), 7.12 (d, J = 1.4 Hz, 1H), 6.14 (s, 1H), 3.80 (s, 1H). 13 13C NMR (150 MHz, Chloroform-d) δ 176.78, 170.26, 164.87, 163.56, 153.86, 153.68, 147.34, 137.51, 136.90, 126.61, 126.25, 125.46, 116.72, 113.51, 111.88, 111.44, 108.68, 102.40, 53.09, 37.07.

[0302] 27. Methyl 2-(pyrido[3,4-b]pyridine-2-carboxylate)-7-(4-bromothiophene-3-carboxylate)coumarin (4f)

[0303] White solid, yield 33.4%. 1 1H NMR (600 MHz, Chloroform-d) δ 8.13 (d, J = 1.6 Hz, 1H), 7.94–7.86 (m, 2H), 7.51 (d, J = 1.6 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.8, 2.4 Hz, 1H), 7.05 (s, 1H), 3.80 (s, 3H). 13 13C NMR (150 MHz, Chloroform-d) δ 176.78, 164.87, 163.56, 161.64, 154.38, 153.79, 147.34, 137.51, 132.92, 128.06, 126.44, 126.41, 116.29, 112.72, 111.62, 111.44, 108.24, 102.40, 53.09.

[0304] 28. Methyl 2-(pyrido[3,4-b]pyridine-2-carboxylate)-7-(3-chlorothiophene-2-carboxylate)coumarin (4g)

[0305] White solid, yield 25.1%. 11H NMR (600 MHz, Chloroform-d) δ 7.94–7.86 (m, 2H), 7.67 (d, J = 4.8 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.8, 2.4 Hz, 1H), 7.13 (d, J = 4.8 Hz, 1H), 7.05 (s, 1H), 3.80 (s, 3H). 13 13C NMR (150 MHz, Chloroform-d) δ 176.78, 164.87, 163.56, 162.90, 153.78, 153.46, 147.34, 137.51, 132.99, 132.18, 131.25, 129.19, 126.43, 116.30, 111.61, 111.44, 108.25, 102.40, 53.09.

[0306] 29. Methyl 2-(pyrido[3,4-b]pyridine-2-carboxylate)-7-(4-bromothiophene-3-carboxylate)coumarin (4h)

[0307] Pale yellow solid, yield 26.4%. 1 1H NMR (500 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.91–7.85 (m, 2H), 7.66 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.8, 2.4 Hz, 1H), 7.05 (s, 1H), 3.80 (s, 3H). 13 13C NMR (150 MHz, Chloroform-d) δ 176.78, 164.87, 163.56, 160.07, 154.10, 153.78, 147.34, 137.51, 134.81, 133.19, 131.27, 126.44, 116.27, 113.09, 111.61, 111.44, 108.50, 102.40, 53.09.

[0308] 30. Methyl 2-(pyrido[3,4-b]pyridine-2-carboxylate)-7-(5-methyl-1,2-oxazole-4-carboxylate)coumarin (4i)

[0309] White solid, yield 35.7%. 11H NMR (500 MHz, Chloroform-d) δ 8.61 (s, 1H), 7.94–7.86 (m, 2H), 7.39 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.8, 2.4 Hz, 1H), 7.05 (s, 1H), 3.80 (s, 3H), 2.72 (s, 3H). 13 13C NMR (150 MHz, Chloroform-d) δ 176.78, 168.07, 164.87, 163.56, 163.21, 154.43, 153.78, 152.60, 147.07, 137.51, 126.44, 116.31, 114.72, 111.86, 111.44, 108.24, 102.40, 53.09, 12.22.

[0310] Example 31

[0311] Performance test:

[0312] 1. Cell culture

[0313] Cell resuscitation: Take out the cryopreserved KYSE-70 cells and KYSE-150 cells from the liquid nitrogen tank, and place them in a constant temperature water bath and shake continuously. After the serum-free cryopreservation solution is completely melted, spray alcohol and transfer them to a biosafety cabinet and place them in a centrifuge tube containing 3 mL of high-glucose DMEM medium containing 10% FBS. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of complete medium and slowly pipette the cells until they are completely suspended. Supplement with 3 mL of complete medium and mix well, then transfer them to a culture flask and culture them in a 37 °C, 5% CO2 cell incubator. When the cell confluence reaches about 90%, they can be taken out for digestion and passage for subsequent experiments.

[0314] Cell culture and passage: When the cells grow to a density of about 90%, spray alcohol on them and transfer them to the cell operation table. Discard the previous medium, suck 3 mL of normal temperature PBS with a Pasteur pipette to wash the cells 2 times, add 1 mL of trypsin to digest the cells for about 3 min, observe under a microscope. When the cell gaps become larger, discard the trypsin. Add an appropriate amount of complete medium to terminate the digestion, pipette the adherent cells down with a pipette, add complete medium to make a single cell suspension, divide it into 2-3 new culture flasks, and continue to culture in a cell incubator. Observe once a day. When the cell confluence reaches about 90%, they can be taken out for digestion and passage for subsequent experiments.

[0315] Cell cryopreservation: When the cell density reaches about 85%, if the cell state is good under the microscope, cryopreservation can be considered. Discard the old culture medium, wash twice with PBS at 37°C, aspirate the residual PBS, add 1 mL of trypsin and digest for about 3 minutes. At the same time, observe the cell morphology under the microscope. When the cells become spherical, immediately transfer them to the operating table, use complete medium to terminate the digestion, gently pipette the cells until they are completely dispersed in the medium, then transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, pour out the upper layer of the medium, use a pipette to aspirate 1 mL of cryopreservation solution into the centrifuge tube, pipette the cells until they are completely suspended in the serum-free cryopreservation solution, then transfer them to a cryopreservation tube. After labeling the cell-related information, place the cryopreservation tube in a ultra-low temperature refrigerator for two days and then transfer it to a liquid nitrogen tank, and make a record.

[0316] In vitro cytotoxicity studies were performed on KYSE-70 cells and KYSE-150 cells by the MTT method. Cells were seeded in 96-well plates with 100 μL of medium and cultured for 24 h. The compounds prepared in all examples were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and then diluted with DMEM to a final concentration of 40 μg / mL. After adding the compounds to the 96-well plates and culturing the cells for 24 h, 10 μL of MTT (5 mg / mL) was added to each well. After culturing the cells for an appropriate time, the absorbance (490 nm) was measured using a microplate spectrophotometer. All experiments were conducted with DMSO (0.5%) as the control.

[0317] The cell survival rate was calculated according to the following formula for the above tests:

[0318] Cell survival rate (%) = (As - Ab) / (Ac - Ab) × 100%

[0319] (As: Absorbance of the experimental well; Ab: Absorbance of the blank well; Ac: Absorbance of the control well)

[0320] The experiment was technically repeated three times, and the results were calculated as the average value.

[0321] After discovering compounds with good activity through primary screening, further study was conducted on the half-inhibitory concentration IC 50 value of the measured drug. Seven concentrations were prepared by the equal gradient dilution method, and the results were measured by the MTT method and calculated using SPSS Statistics 27.0.

[0322] 2. Results and Analysis

[0323] The in vitro anti-tumor activity experiment was carried out by the MTT method, using the synthesized pyridone-coumarin derivatives as the active objects against human esophageal squamous cell carcinoma cells KYSE-70 and KYSE-150. The 48-hour cell survival rate is shown in Table 1. It was found that they had certain anti-tumor activity at a concentration of 40 μg / mL.

[0324] Table 1 Cell viability of coumarin derivatives on KYSE-70 and KYSE-150 after 48h

[0325]

[0326]

[0327] From the results in the above table, it can be seen that the pyridone-type coumarin derivatives synthesized in the present invention have a certain inhibitory effect on both KYSE-70 and KYSE-150. In particular, compounds 1, 1d, 2, 2e, 2h, 3, 3b, 3c, and 4a have better inhibitory effects on KYSE-70 and KYSE-150. Among them, the inhibition rate (inhibition rate = 100% - cell viability) of compound 4a on KYSE-70 at 40 μg / mL is about 90.8%, and the inhibition rate on KYSE-150 is 73.35%. Further, when the concentration is reduced to 10 μg / mL, the inhibition rate on KYSE-70 is still about 85.88%.

[0328] Due to the outstanding effects of compound 4a on KYSE-70 and KYSE-150, its IC 50 values are further studied as shown in Table 2. Seven concentrations are obtained by the equal gradient dilution method, and then through SPPS calculation, the IC 50 of compound 4a on KYSE-70 is 3.38 μg / mL, and the IC 50 on KYSE-150 is 18.95 μg / mL.

[0329] Table 2 IC 50

[0330]

[0331] From the above results, it can be seen that the pyridone-type coumarin derivatives of the present invention have good application value in anti-inflammatory, anti-viral, antibacterial or anti-cancer.

[0332] The present invention has been described in detail above in combination with specific embodiments and exemplary examples, but these descriptions should not be construed as limiting the present invention. Those skilled in the art understand that without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements can be made to the technical solutions and their implementation manners of the present invention, and these all fall within the scope of the present invention. The protection scope of the present invention is subject to the appended claims.

[0333] All publications, patent applications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. In case of conflict, the definitions in this specification shall prevail.

[0334] When this specification uses the prefixes "known to those skilled in the art", "prior art", or similar terms to introduce materials, substances, methods, steps, devices, or components, etc., the objects introduced by such prefixes cover those commonly used in the art at the time of filing of this application, but also include those that are not commonly used at present but will become recognized in the art as suitable for similar purposes.

[0335] In the context of this specification, any matter or thing not mentioned, other than what is expressly stated, shall directly apply those known in the art without any change.

Claims

1. A pyridone-type coumarin derivative, characterized in that, The pyridone-coumarin derivatives are selected from the following general structural formulas: Among them, R1 in Formula 1 is selected from hydrogen, a substituted or unsubstituted carbonyl group; Among them, R2 in Formula 2 is selected from hydrogen, a substituted or unsubstituted carbonyl group; Among them, R3 in Formula 3 is selected from hydrogen, a substituted or unsubstituted carbonyl group; Among them, R4 in Formula 4 is selected from a hydroxyl group, a substituted alkoxy group, a substituted ester group; R5 is selected from hydrogen, an alkyl group.

2. The pyridone-coumarin derivative according to claim 1, characterized in that: R1 in the formula 1 is selected from hydrogen, wherein, R 1a is selected from alkenyl, substituted or unsubstituted phenyl; Preferably, R 1a is selected from alkenyl having 2 to 8 carbon atoms; halogen, phenyl or phenyl substituted with at least one alkyl having 1 to 4 carbon atoms; Further preferably, R 1a is selected from a linear alkenyl group having 2 to 6 carbon atoms, a phenyl group with two halogen atoms in the meta position, a phenyl group with one halogen atom and one C1-C2 alkyl group in the meta position, and a phenyl group; More preferably, Formula 1 is selected from the following compounds:

3. The pyridone-coumarin derivative according to claim 1, characterized in that: R2 in the formula 2 is selected from hydrogen, wherein, R 2a is selected from alkenyl, substituted or unsubstituted phenyl; Preferably, R 2a An alkenyl group having 2 to 8 carbon atoms; a phenyl group substituted with at least one of a halogen, an alkyl group having 1 to 4 carbon atoms, a fluoroalkyl group, or a hydroxyl group; a phenyl group; Further preferably, R 2a is selected from alkenyl having 3 to 8 carbon atoms, phenyl substituted by two halogens in the meta position, phenyl substituted by one halogen and one C1-C2 alkyl group, phenyl substituted by three halogens, phenyl substituted by one fluoroalkyl group, phenyl substituted by one fluoroalkyl group and one halogen in the ortho position, phenyl substituted by one fluoroalkyl group and one hydroxyl group in the meta position, phenyl; More preferably, Formula 2 is selected from the following compounds:

4. The pyridone-coumarin derivative according to claim 1, characterized in that: R3 in the formula 3 is selected from hydrogen, wherein, R 3a is selected from substituted or unsubstituted phenyl; substituted or unsubstituted thienyl; Preferably, R 3a selected from fluoroalkyl-substituted phenyl, phenyl, and thiophenyl; Further preferably, R 3a is selected from a fluoroalkyl-substituted phenyl, phenyl, and thiophenyl; More preferably, Formula 3 is selected from the following compounds:

5. The pyridone-coumarin derivative according to claim 1, characterized in that: R4 in the formula 4 is selected from hydrogen, wherein, R 4a1 is selected from pyridyl with substituents; R 4a2 is selected from C3-C6 cycloalkyl, halogen-substituted thienyl, C1-C4 alkyl-substituted oxazolyl; R5 is selected from hydrogen, an alkyl group having 1 to 4 carbon atoms; Preferably, R 4a1 is selected from pyridyl groups containing an alkylcarbonyl substituent; R 4a2 is selected from C3-C4 cycloalkyl groups, a halogen-substituted thiophenyl group, a C1-C2 alkyl-substituted oxazolyl group; and / or, R5 is selected from hydrogen, an alkyl group having 1 to 2 carbon atoms; More preferably, Formula 4 is selected from the following compounds:

6. A method for preparing a pyridone-based coumarin derivative according to any one of claims 1-5, characterized in that, Comprising the following steps: Method 1: React compound 1 with R 1a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 1a -H in R 1a is the same as the R 1a corresponding to any one of claims 1-5; Among them, the structural formula of Compound 1 is: Method 2: React compound 2 with R 2a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 2a -H in which R 2a is the same as the R 2a corresponding to any one of claims 1-5; Among them, the structural formula of Compound 2 is: Method 3: React compound 3 with R 3a -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 3a -H in R 3a is the same as the R 3a corresponding to any one of claims 1-5; Among them, the structural formula of Compound 3 is: Method Four: Step (1) Under a protective atmosphere, Compound 4 and a cyclization catalyst are heated and reacted, followed by post-treatment to obtain Compound 4a; Optional step (2): React Compound 4a with an alkali solution to obtain the pyridone-coumarin derivative; Optional step (3): React compound 4a with R 4a1 -X, a nucleophile, and a base scavenger in a solvent to obtain the pyridone-type coumarin derivative; R 4a1 in R 4a1 is the same as the R 4a1 corresponding to any one of claims 1-5; X is a halogen; Optional step (4): React compound 4a with R 4a2 -H, a condensing agent, and a basic catalyst in a solvent to obtain the pyridone-type coumarin derivative; R 4a2 -H in which R 4a2 is the same as the R 4a2 corresponding to any one of claims 1-5; Among them, the structural formula of Compound 4 is: The structural formula of Compound 4a is:

7. The preparation method of the pyridone-coumarin derivative according to claim 6, characterized in that: In Method One, Method Two, Method Three, and step (4) of Method Four, the condensing agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or N,N-dicyclohexylcarbodiimide; and / or, In Method One, Method Two, Method Three, and step (4) of Method Four, the basic catalyst is selected from 4-dimethylaminopyridine; and / or, In Method One, Method Two, Method Three, and step (4) of Method Four, the solvent is selected from at least one of dichloromethane, DMF, ether, or tetrahydrofuran; and / or, In Method Four, the cyclization catalyst in step (1) is selected from at least one of polyphosphoric acid or Eaton's reagent; and / or, In step (2), the base in the alkali solution is selected from inorganic bases, preferably selected from at least one of NaOH, sodium carbonate, or sodium bicarbonate; and / or, In step (3), the nucleophile is selected from halogen salts, preferably selected from at least one of KI or NaI; and / or, In step (3), the acid-binding agent is selected from at least one of potassium carbonate, sodium carbonate, or sodium bicarbonate; and / or, In step (3), the solvent is selected from at least one of DMF, dichloromethane, or ether.

8. The preparation method of the pyridone-coumarin derivative according to claim 6, characterized in that: In step (4) of Method One, Method Two, Method Three, and Method Four, The molar ratio of the condensing agent to the basic catalyst is independently (5-8):1; and / or, The mass-volume ratio of the condensing agent to the solvent is independently (2-20) mg: 1 mL; and / or, In Method 1, the molar ratio of Compound 1 to R 1a -H is 1:(2 to 4); and / or, The molar ratio of Compound 1 to the condensing agent is 1: (2-6); and / or, In Method 2, the molar ratio of Compound 2 to R 2a -H is 1:(2 - 4); and / or, The molar ratio of Compound 2 to the condensing agent is 1: (2-6); and / or, In Method 3, the molar ratio of Compound 3 to R 3a -H is 1:(2 - 4); and / or, The molar ratio of Compound 3 to the condensing agent is 1: (2-6); and / or, In step (4) of Method 4, the molar ratio of compound 4a to R 4a2 -H is 1:(1 to 4); and / or, The molar ratio of Compound 4a to the condensing agent is 1: (1-3); and / or, In Method 1, Method 2, Method 3, and Method 4, the reaction time in step (4) is independently 5-10 h; and / or, In Method 4, In step (1), the mass ratio of Compound 4 to the cyclization catalyst is 1: (40-60); and / or, In step (1), the temperature of the heating reaction is 120-160 °C; and / or, In step (1), the heating reaction time is 4-5 h; and / or, In step (2), after adding the alkali solution, the pH value of the reaction system is 6-7; and / or, The reaction time of step (2) is 2-4 h; and / or, Step (3), the molar ratio of compound 4a to R 4a1 -X is 1:(1-2); and / or, In step (3), the molar ratio of Compound 4a to the nucleophile is 1: (1-2); and / or, In step (3), the molar volume ratio of Compound 4a to the solvent is 1 mmol: (20-60) mL; and / or, In step (3), the molar ratio of Compound 4a to the acid-binding agent is 1: (6-9); and / or, The reaction time of step (3) is 2-8 h.

9. Use of a pyridone-coumarin derivative according to any one of claims 1-5 in at least one of anti-inflammatory, anti-viral, antibacterial, or anti-cancer.

10. The use according to claim 9, characterized in that: The use of the pyridone-coumarin derivative in inhibiting KYSE-70 and / or KYSE-150 cells.

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